Production of fatty acid derivatives

ABSTRACT

The invention relates to compositions and methods, including polynucleotide sequences, amino acid sequences, recombinant host cells and recombinant host cell cultures engineered to produce fatty acid derivative compositions comprising fatty acids, fatty alcohols, fatty aldehydes, fatty esters, alkanes, terminal olefins, internal olefins or ketones. The fatty acid derivative composition is produced extracellularly with a higher titer, yield or productivity than the corresponding wild type or non-engineered host cell.

FIELD OF THE INVENTION

The invention relates to engineered host cells together with vector and strain modifications effective to improve the titer, yield and productivity of fatty acid derivatives relative to “wild-type” or non-engineered host cells. The invention further relates to methods of making and using such modified vectors and strains for the fermentative production of fatty acid derivatives and fatty acid derivative compositions.

BACKGROUND OF THE INVENTION

Fatty acid derivatives including fatty aldehydes, fatty alcohols, hydrocarbons (alkanes and olefins), fatty esters (e.g., waxes, fatty acid esters, or fatty esters) and ketones comprise important categories of industrial chemicals and fuels. These molecules and their derivatives have numerous applications including use as surfactants, lubricants, plasticizers, solvents, emulsifiers, emollients, thickeners, flavors, fragrances, and fuels.

Crude petroleum is currently a primary source of raw materials for producing petrochemicals and fuels. The two main classes of raw materials derived from petroleum are short chain olefins (e.g., ethylene and propylene) and aromatics (e.g., benzene and xylene isomers). These raw materials are derived from longer chain hydrocarbons in crude petroleum by cracking it at considerable expense using a variety of methods, such as catalytic cracking, steam cracking, or catalytic reforming. These raw materials can be used to make petrochemicals such as monomers, solvents, detergents, and adhesives, which otherwise cannot be directly refined from crude petroleum.

Petrochemicals, in turn, can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, and gels. Particular specialty chemicals that can be produced from petrochemical raw materials include fatty acids, hydrocarbons, fatty aldehydes, fatty alcohols, esters, ketones, etc.

Hydrocarbons have many commercial uses. For example, shorter chain alkanes and alkenes are used in transportation fuels. Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants. In addition, alkenes are used as a feedstock to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinic anhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives, and drag flow reducers.

Esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.

Aldehydes are used to produce many specialty chemicals. For example, aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which may be used as solvents, preservatives, or disinfectants. In addition, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction.

Fatty alcohols have many commercial uses. The shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, such as, for example, detergents. In addition, fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats.

Fatty alcohols are aliphatic alcohols consisting of a chain of 8 to 22 carbon atoms. Fatty alcohols usually have even number of carbon atoms and a single alcohol group (—OH) attached to the terminal carbon. Some are unsaturated and some are branched. They are widely used in industrial chemistry. Most fatty alcohols in nature are found as waxes which are esters with fatty acids and fatty alcohols. They are produced by bacteria, plants and animals.

Currently, fatty alcohols are produced via catalytic hydrogenation of fatty acids produced from natural sources, such as coconut oil, palm oil, palm kernel oil, tallow and lard, or by chemical hydration of alpha-olefins produced from petrochemical feedstocks. Fatty alcohols derived from natural sources have varying chain lengths. The chain length of fatty alcohols is important and specific to particular applications. Dehydration of fatty alcohols to alpha-olefins can also be accomplished by chemical catalysis.

Due to the inherent challenges posed by exploring, extracting, transporting and refining petroleum for use in chemical and fuel products, there is a need for a an alternate source which can be produced economically and used for chemical and fuel production. There is also a need for a petroleum replacement that does not cause the type of environmental damage created by the exploring, extracting, transporting and refining petroleum and the burning of petroleum-based fuels.

One method of producing renewable petroleum is by engineering host cells to produce renewable petroleum products. Biologically derived fuels and chemicals offer advantages over petroleum based fuels. Biologically derived chemicals such as hydrocarbons (e.g., alkanes, alkenes, or alkynes), fatty alcohols, esters, fatty acids, fatty aldehydes, and ketones are directly converted from biomass to the desired chemical product.

In order for the use of biologically-derived fatty acid derivatives from fermentable sugars or biomass to be commercially viable as a source for production of renewable chemicals and fuels, the process must be optimized for efficient conversion and recovery of product. The development of biologically derived fuels and chemicals has been a focus of research and development in recent years, however, there remains a need for improvement in the relevant processes and products in order for biologically derived fuels and chemicals to become a commercially viable option. Areas for improvement include the energy efficiency of the production process and product yield. The current invention addresses this need.

SUMMARY OF THE INVENTION

The present invention provides novel genetically engineered host cells which produce fatty acid derivative compositions at a high titer, yield or productivity; cell cultures comprising such novel genetically engineered host cells and methods of using the same. The invention also provides methods of making compositions comprising fatty acid derivatives by culturing the genetically engineered host cells of the invention, compositions made by such methods, and other features apparent upon further review.

In one embodiment, the invention provides a cultured genetically engineered host cell comprising (a) a polynucleotide sequence encoding one or more of: (i) an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, (iv) a sequence having a transposon insertion in the yijP gene, and (v) a heterologous ACP protein; as well as (b) a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide, wherein the genetically engineered host cell produces a fatty acid derivative composition at a higher titer, yield or productivity when cultured in medium containing a carbon source under conditions effective to overexpress the polynucleotide(s) relative to a corresponding wild type host cell propagated under the same conditions as the genetically engineered host cell.

The fatty acid derivative composition includes one or more of a fatty acid, a fatty aldehyde, a fatty alcohol, a fatty ester, an alkane, a terminal olefin, an internal olefin and a ketone.

In one embodiment, the genetically engineered host cell produces a fatty acid derivative composition with a titer, yield or productivity that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the titer of a fatty acid derivative composition produced by a corresponding wild type (non-engineered) host cell propagated under the same conditions as the genetically engineered host cell (e.g., a titer of from 30 g/L to 250 g/L, a yield of from 10% to 40%, or a productivity of 0.7 mg/L/hr to 3 g/L/hr).

In some embodiments, the fatty acid derivative composition is produced extracellularly.

In other embodiments, the host cell is further engineered to comprise a heterologous acp sequence with or without an introduced sfp gene.

The polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide is selected from the group consisting of a polypeptide:

(a) having thioesterase activity, wherein the recombinant host cell synthesizes fatty acids;

(b) having thioesterase activity and carboxylic acid reductase (“CAR”) activity, wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols;

(c) having thioesterase activity, carboxylic acid reductase activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols;

(d) having acyl-CoA reductase (“AAR”) activity wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols;

(e) having acyl-CoA reductase (“AAR”) activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols;

(f) having fatty alcohol forming acyl-CoA reductase (“FAR”) activity, wherein the recombinant host cell synthesizes fatty alcohols;

(g) having thioesterase activity, carboxylic acid reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes;

(h) having acyl-CoA reductase (“AAR”) activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes;

(i) having ester synthase activity wherein the recombinant host cell synthesizes fatty esters;

(j) having thioesterase activity, acyl-CoA synthase activity and ester synthase activity wherein the recombinant host cell synthesizes fatty esters;

(k) having OleA activity, wherein the recombinant host cell synthesizes aliphatic ketones;

(l) having OleABCD activity, wherein the recombinant host cell synthesizes internal olefins; and

(m) having thioesterase activity and decarboxylase activity, wherein the recombinant host cell synthesizes terminal olefins.

These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents an exemplary biosynthetic pathway for use in production of acyl CoA as a precursor to fatty acid derivatives in a recombinant microorganism. The cycle is initiated by condensation of malonyl-ACP and acetyl-CoA.

FIG. 2 presents an exemplary fatty acid biosynthetic cycle, where malonyl-ACP is produced by the transacylation of malonyl-CoA to malonyl-ACP (catalyzed by malonyl-CoA:ACP transacylase; fabD), then β-ketoacyl-ACP synthase III (fabH) initiates condensation of malonyl-ACP with acetyl-CoA. Elongation cycles begin with the condensation of malonyl-ACP and an acyl-ACP catalyzed by β-ketoacyl-ACP synthase I (fabB) and β-ketoacyl-ACP synthase II (fabF) to produce a β-keto-acyl-ACP, then the β-keto-acyl-ACP is reduced by β-ketoacyl-ACP reductase (fabG) to produce a β-hydroxy-acyl-ACP, which is dehydrated to a trans-2-enoyl-acyl-ACP by β-hydroxyacyl-ACP dehydratase (fabA or fabZ). FabA can also isomerize trans-2-enoyl-acyl-ACP to cis-3-enoyl-acyl-ACP, which can bypass fabI and can used by fabB (typically for up to an aliphatic chain length of C16) to produce β-keto-acyl-ACP. The final step in each cycle is catalyzed by enoyl-ACP reductase (fabI) that converts trans-2-enoyl-acyl-ACP to acyl-ACP. In the methods described herein, termination of fatty acid synthesis occurs by thioesterase removal of the acyl group from acyl-ACP to release free fatty acids (FFA). Thioesterases (e.g., tesA) hydrolyze thioester bonds, which occur between acyl chains and ACP through sulfhydryl bonds.

FIG. 3 illustrates the structure and function of the acetyl-CoA carboxylase (accABCD) enzyme complex.

FIG. 4 presents an overview of an exemplary biosynthetic pathway for production of fatty alcohol starting with acyl-ACP, where the production of fatty aldehyde is catalyzed by the enzymatic activity of acyl-ACP reductase (AAR) or thioesterase and carboxylic acid reductase (Car). The fatty aldehyde is converted to fatty alcohol by aldehyde reductase (also referred to as alcohol dehydrogenase).

FIG. 5 presents an overview of two exemplary biosynthetic pathways for production of fatty esters starting with acyl-ACP, where the production of fatty esters is accomplished by a one enzyme system or a three enzyme system.

FIG. 6 presents an overview of exemplary biosynthetic pathways for production of hydrocarbons starting with acyl-ACP, where the production of ketones is catalyzed by the enzymatic activity of OleA; the production of internal olefins is catalyzed by the enzymatic activity of OleABCD; the production of alkanes is catalyzed by the enzymatic conversion of fatty aldehydes to alkanes by way of aldehyde decarbonylase to; and the production of terminal olefins is catalyzed by the enzymatic conversion of fatty acids to terminal olefins by a decarboxylase

FIG. 7 illustrates fatty acid derivative (“Total Fatty Species”) production by the MG1655 E. coli strain with the fadE gene attenuated (i.e., deleted) compared to fatty acid derivative production by E. coli MG1655. The data presented in FIG. 7 shows that attenuation of the fadE gene did not affect fatty acid derivative production.

FIG. 8 shows malonyl-CoA levels in DAM1_i377 in log phase expressing eight different C. glutamicum acetyl-CoA carboxylase (Acc) operon constructs.

FIG. 9 shows intracellular short chain-CoA levels in E. coli DAM1_(—±377) in log phase expressing ptrc1/3_accDACB-birA±panK operon constructs. “accDACB+birA” is also referred to herein as “accD+”.

FIG. 10 shows fatty acid methyl ester (FAME) production in E. coli strain DV2 expressing ester synthase 9 from M. hydrocarbonoclasticus and components of an acetyl-CoA carboxylase complex from C. glutamicum.

FIG. 11 shows production of fatty alcohols by E. coli expressing the Synechococcus elongatus PCC7942 AAR together with the accD+operon” from C. glutamicum on a pCL plasmid. Triplicate samples are shown for the accD+strains.

FIGS. 12A and B show data for production of “Total Fatty Species” (mg/L) from duplicate plate screens when plasmid pCL-WT TRC WT TesA was transformed into each of the iFAB-containing strains shown in the figures and a fermentation was run in FA2 media with 20 hours from induction to harvest at both 32° C. (FIG. 12A) and 37° C. (FIG. 12B).

FIG. 13 shows FAME production of E. coli DAM1 with plasmid pDS57 and integrated fabHI operons. The fabH/I genes are from Marinobacter aquaeoli VT8 or from Acinetobacter baylyi ADP1. See Table 7 for a more details on the fabH/I operons in these strains.

FIG. 14 shows FAME production of E. coli DAM 1 with plasmid pDS57 and different configurations of the C. glutamicum acc genes as well as integrated fabHI operons. The strains contain the fabH/I genes from Rhodococcus opacus or Acinetobacter baylyi ADP1. See Table 7 for more details on the fabH/I and acc operons.

FIG. 15 shows FAME and FFA titers of two E. coli DAM1 pDS57 strains with integrated fabH/I genes strains selected from FIG. 13 compared to the control strain E. coli DAM1 pDS57.

FIG. 16 is a diagrammatic depiction of the iFAB 138 locus, including a diagram of cat-loxP-T5 promoter integrated in front of FAB 138 (16A); and a diagram of iT5_(—)138 (16B). The sequence of cat-loxP-T5 promoter integrated in front of FAB138 with 50 base pair of homology shown on each side of cat-loxP-T5 promoter region is provided as SEQ ID NO:1 and the sequence of the iT5_(—)138 promoter region with 50 base pair homology on each side is provided as SEQ ID NO:2.

FIG. 17 shows that correcting the rph and ilvG genes in the EG149 strain allows for a higher level of FFA production than in the V668 strain where the rph and ilvG genes were not corrected.

FIG. 18 is a diagrammatic depiction of a transposon cassette insertion in the yijP gene of strain LC535 (transposon hit 68F11). Promoters internal to the transposon cassette are shown, and may have effects on adjacent gene expression.

FIG. 19 illustrates fatty alcohol production in E. coli DV2 expressing Synechococcus elongatus acyl-ACP reductase (AAR) and coexpressing various cyanobacterial acyl carrier proteins (ACPs). (Details regarding the source of the ACPs are provided in Table 13).

FIG. 20 illustrates fatty acid production in E. coli DV2 expressing leaderless E. coli thioesterase 'tesA and coexpressing a cyanobacterial acyl carrier protein (cACP) and B. subtilis sfp.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, at least in part, on the discovery that modification of various aspects of the fatty acid biosynthetic pathway in a recombinant host cell facilitates enhanced production of fatty acid derivatives by the host cell.

The invention relates to compositions of fatty acid derivatives having desired characteristics and methods for producing the same. Further, the invention relates to recombinant host cells (e.g., microorganisms), cultures of recombinant host cells, methods of making and using recombinant host cells, for example, use of cultured recombinant host cells in the fermentative production of fatty acid derivatives having desired characteristics.

All patents, publications, and patent applications cited in this specification are herein incorporated by reference as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

DEFINITIONS

As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a recombinant host cell” includes two or more such recombinant host cells, reference to “a fatty alcohol” includes one or more fatty alcohols, or mixtures of fatty alcohols, reference to “a nucleic acid coding sequence” includes one or more nucleic acid coding sequences, reference to “an enzyme” includes one or more enzymes, and the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although other methods and materials similar, or equivalent, to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

Accession Numbers: Sequence Accession numbers throughout this description were obtained from databases provided by the NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health, U.S.A. (which are identified herein as “NCBI Accession Numbers” or alternatively as “GenBank Accession Numbers”), and from the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Institute of Bioinformatics (which are identified herein as “UniProtKB Accession Numbers”).

Enzyme Classification (EC) Numbers: EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), description of which is available on the IUBMB Enzyme Nomenclature website on the World Wide Web. EC numbers classify enzymes according to the reaction catalyzed.

As used herein, the term “nucleotide” refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups. The naturally occurring bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typically derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleic acids are typically linked via phosphate bonds to form nucleic acids or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).

As used herein, the term “polynucleotide” refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms “polynucleotide,” “nucleic acid sequence,” and “nucleotide sequence” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either RNA or DNA. These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to methylated and/or capped polynucleotides. The polynucleotide can be in any form, including but not limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.

As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide.

As used herein, the terms “homolog,” and “homologous” refer to a polynucleotide or a polypeptide comprising a sequence that is at least about 50% identical to the corresponding polynucleotide or polypeptide sequence. Preferably homologous polynucleotides or polypeptides have polynucleotide sequences or amino acid sequences that have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% homology to the corresponding amino acid sequence or polynucleotide sequence. As used herein the terms sequence “homology” and sequence “identity” are used interchangeably.

One of ordinary skill in the art is well aware of methods to determine homology between two or more sequences. Briefly, calculations of “homology” between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a first sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 90%, or about 100% of the length of a second sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions of the first and second sequences are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent homology between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, that need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm, such as BLAST (Altschul et al., J. Mol. Biol., 215(3): 403-410 (1990)). The percent homology between two amino acid sequences also can be determined using the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol., 48: 444-453 (1970)). The percent homology between two nucleotide sequences also can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in the art can perform initial homology calculations and adjust the algorithm parameters accordingly. A preferred set of parameters (and the one that should be used if a practitioner is uncertain about which parameters should be applied to determine if a molecule is within a homology limitation of the claims) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the biotechnology arts (see, e.g., Rosenberg, BMC Bioinformatics, 6: 278 (2005); Altschul, et al., FEBS J., 272(20): 5101-5109 (2005)).

As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either method can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions—6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions—6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions—6×SSC at about 45° C., followed by one or more washes in 0.2.×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridization conditions—0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.

An “endogenous” polypeptide refers to a polypeptide encoded by the genome of the parental microbial cell (also termed “host cell”) from which the recombinant cell is engineered (or “derived”).

An “exogenous” polypeptide refers to a polypeptide which is not encoded by the genome of the parental microbial cell. A variant (i.e., mutant) polypeptide is an example of an exogenous polypeptide.

The term “heterologous” as used herein typically refers to a nucleotide sequence or a protein not naturally present in an organism. For example, a polynucleotide sequence endogenous to a plant can be introduced into a host cell by recombinant methods, and the plant polynucleotide is then a heterologous polynucleotide in a recombinant host cell.

As used herein, the term “fragment” of a polypeptide refers to a shorter portion of a full-length polypeptide or protein ranging in size from four amino acid residues to the entire amino acid sequence minus one amino acid residue. In certain embodiments of the invention, a fragment refers to the entire amino acid sequence of a domain of a polypeptide or protein (e.g., a substrate binding domain or a catalytic domain).

As used herein, the term “mutagenesis” refers to a process by which the genetic information of an organism is changed in a stable manner. Mutagenesis of a protein coding nucleic acid sequence produces a mutant protein. Mutagenesis also refers to changes in non-coding nucleic acid sequences that result in modified protein activity.

As used herein, the term “gene” refers to nucleic acid sequences encoding either an RNA product or a protein product, as well as operably-linked nucleic acid sequences affecting the expression of the RNA or protein (e.g., such sequences include but are not limited to promoter or enhancer sequences) or operably-linked nucleic acid sequences encoding sequences that affect the expression of the RNA or protein (e.g., such sequences include but are not limited to ribosome binding sites or translational control sequences).

Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

In the methods of the invention, an expression control sequence is operably linked to a polynucleotide sequence. By “operably linked” is meant that a polynucleotide sequence and an expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s). Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.

As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it has been linked One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal replication). Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. The terms “plasmid” and “vector” are used interchangeably herein, in as much as a plasmid is the most commonly used form of vector. However, also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.

In some embodiments, a recombinant vector further comprises a promoter operably linked to the polynucleotide sequence. In some embodiments, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter. The recombinant vector typically comprises at least one sequence selected from the group consisting of (a) an expression control sequence operatively coupled to the polynucleotide sequence; (b) a selection marker operatively coupled to the polynucleotide sequence; (c) a marker sequence operatively coupled to the polynucleotide sequence; (d) a purification moiety operatively coupled to the polynucleotide sequence; (e) a secretion sequence operatively coupled to the polynucleotide sequence; and (f) a targeting sequence operatively coupled to the polynucleotide sequence. In certain embodiments, the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region.

The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein. Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy-terminus of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. In certain embodiments, a polynucleotide sequence of the invention is operably linked to a promoter derived from bacteriophage T5.

In certain embodiments, the host cell is a yeast cell, and the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., EMBO J., 6: 229-234 (1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultz et al., Gene, 54: 113-123 (1987)), pYES2 (Invitrogen Corp., San Diego, Calif.), and picZ (Invitrogen Corp., San Diego, Calif.).

In other embodiments, the host cell is an insect cell, and the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include, for example, the pAc series (Smith et al., Mol. Cell. Biol., 3: 2156-2165 (1983)) and the pVL series (Lucklow et al., Virology, 170: 31-39 (1989)).

In yet another embodiment, the polynucleotide sequences described herein can be expressed in mammalian cells using a mammalian expression vector. Other suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” second edition, Cold Spring Harbor Laboratory, (1989).

As used herein “acyl-CoA” refers to an acyl thioester formed between the carbonyl carbon of alkyl chain and the sulfhydryl group of the 4′-phosphopantethionyl moiety of coenzyme A (CoA), which has the formula R—C(O)S—CoA, where R is any alkyl group having at least 4 carbon atoms.

As used herein “acyl-ACP” refers to an acyl thioester formed between the carbonyl carbon of alkyl chain and the sulfhydryl group of the phosphopantetheinyl moiety of an acyl carrier protein (ACP). The phosphopantetheinyl moiety is post-translationally attached to a conserved serine residue on the ACP by the action of holo-acyl carrier protein synthase (ACPS), a phosphopantetheinyl transferase. In some embodiments an acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACPs. In other embodiments an acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACPs. In some embodiments, the carbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons. Each of these acyl-ACPs are substrates for enzymes that convert them to fatty acid derivatives such as those described in FIGS. 4-6

As used herein, the term “fatty acid derivative” means a “fatty acid” or a “fatty acid derivative”, which may be referred to as a “fatty acid or derivative thereof”. The term “fatty acid” means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can comprise between about 4 and about 22 carbon atoms. Fatty acids can be saturated, monounsaturated, or polyunsaturated. A “fatty acid derivative” is a product made in part from the fatty acid biosynthetic pathway of the production host organism. “Fatty acid derivatives” includes products made in part from acyl-ACP or acyl-ACP derivatives. Exemplary fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, fatty alcohols, hydrocarbons, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones.

A “fatty acid derivative composition” as referred to herein is produced by a recombinant host cell and typically comprises a mixture of fatty acid derivative. In some cases, the mixture includes more than one type of product (e.g., fatty acids and fatty alcohols, fatty acids and fatty acid esters or alkanes and olefins). In other cases, the fatty acid derivative compositions may comprise, for example, a mixture of fatty alcohols (or another fatty acid derivative) with various chain lengths and saturation or branching characteristics. In still other cases, the fatty acid derivative composition comprises a mixture of both more than one type of product and products with various chain lengths and saturation or branching characteristics.

As used herein, the term “fatty acid biosynthetic pathway” means a biosynthetic pathway that produces fatty acids and derivatives thereof. The fatty acid biosynthetic pathway may include additional enzymes to produce fatty acids derivatives having desired characteristics.

As used herein, “fatty aldehyde” means an aldehyde having the formula RCHO characterized by a carbonyl group (C═O). In some embodiments, the fatty aldehyde is any aldehyde made from a fatty alcohol. In certain embodiments, the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length. Thus, the R group can have an R group bounded by any two of the above endpoints. For example, the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length. In some embodiments, the fatty aldehyde is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 fatty aldehyde. In certain embodiments, the fatty aldehyde is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 fatty aldehyde.

As used herein, “fatty alcohol” means an alcohol having the formula ROH. In some embodiments, the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length. Thus, the R group can have an R group bounded by any two of the above endpoints. For example, the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length. In some embodiments, the fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 fatty alcohol. In certain embodiments, the fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 fatty alcohol.

The R group of a fatty acid derivative, for example a fatty alcohol, can be a straight chain or a branched chain. Branched chains may have more than one point of branching and may include cyclic branches. In some embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C₂₋₆ branched fatty acid, branched fatty aldehyde, or branched fatty alcohol. In particular embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 branched fatty acid, branched fatty aldehyde, or branched fatty alcohol. In certain embodiments, the hydroxyl group of the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is in the primary (C1) position.

In certain embodiments, the branched fatty acid derivative is an iso-fatty acid derivative, for example an iso-fatty aldehyde, an iso-fatty alcohol, or an antesio-fatty acid derivative, an anteiso-fatty aldehyde, or an anteiso-fatty alcohol. In exemplary embodiments, the branched fatty acid derivative is selected from iso-C7:0, iso-C8:0, iso-C9:0, iso-C10:0, iso-C11:0, iso-C12:0, iso-C13:0, iso-C14:0, iso-C15:0, iso-C16:0, iso-C17:0, iso-C18:0, iso-C19:0, anteiso-C7:0, anteiso-C8:0, anteiso-C9:0, anteiso-C10:0, anteiso-C11:0, anteiso-C12:0, anteiso-C13:0, anteiso-C14:0, anteiso-C15:0, anteiso-C16:0, anteiso-C17:0, anteiso-C18:0, and an anteiso-C19:0 branched fatty alcohol.

The R group of a branched or unbranched fatty acid derivative can be saturated or unsaturated. If unsaturated, the R group can have one or more than one point of unsaturation. In some embodiments, the unsaturated fatty acid derivative is a monounsaturated fatty acid derivative. In certain embodiments, the unsaturated fatty acid derivative is a C6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1 unsaturated fatty acid derivative. In certain embodiments, the unsaturated fatty acid derivative, is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. In other embodiments, the unsaturated fatty acid derivative is unsaturated at the omega-7 position. In certain embodiments, the unsaturated fatty acid derivative comprises a cis double bond.

As used herein, a recombinant or engineered “host cell” is a host cell, e.g., a microorganism used to produce one or more of fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones.

In some embodiments, the recombinant host cell comprises one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty acid biosynthetic enzyme activity, wherein the recombinant host cell produces a fatty acid derivative composition when cultured in the presence of a carbon source under conditions effective to express the polynucleotides.

As used herein, the term “clone” typically refers to a cell or group of cells descended from and essentially genetically identical to a single common ancestor, for example, the bacteria of a cloned bacterial colony arose from a single bacterial cell.

As used herein, the term “culture” typical refers to a liquid media comprising viable cells. In one embodiment, a culture comprises cells reproducing in a predetermined culture media under controlled conditions, for example, a culture of recombinant host cells grown in liquid media comprising a selected carbon source and nitrogen.

“Culturing” or “cultivation” refers to growing a population of recombinant host cells under suitable conditions in a liquid or solid medium. In particular embodiments, culturing refers to the fermentative bioconversion of a substrate to an end-product. Culturing media are well known and individual components of such culture media are available from commercial sources, e.g., under the Difco™ and BBL™ trademarks. In one non-limiting example, the aqueous nutrient medium is a “rich medium” comprising complex sources of nitrogen, salts, and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extract of such a medium.

The host cell can be additionally engineered to assimilate carbon efficiently and use cellulosic materials as carbon sources according to methods described in U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030; WO 2010127318. In addition, in some embodiments the host cell is engineered to express an invertase so that sucrose can be used as a carbon source.

As used herein, the term “under conditions effective to express said heterologous nucleotide sequence(s)” means any conditions that allow a host cell to produce a desired fatty acid derivative. Suitable conditions include, for example, fermentation conditions.

As used herein, “modified” or an “altered level of” activity of a protein, for example an enzyme, in a recombinant host cell refers to a difference in one or more characteristics in the activity determined relative to the parent or native host cell. Typically differences in activity are determined between a recombinant host cell, having modified activity, and the corresponding wild-type host cell (e.g., comparison of a culture of a recombinant host cell relative to the corresponding wild-type host cell). Modified activities can be the result of, for example, modified amounts of protein expressed by a recombinant host cell (e.g., as the result of increased or decreased number of copies of DNA sequences encoding the protein, increased or decreased number of mRNA transcripts encoding the protein, and/or increased or decreased amounts of protein translation of the protein from mRNA); changes in the structure of the protein (e.g., changes to the primary structure, such as, changes to the protein's coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters); and changes in protein stability (e.g., increased or decreased degradation of the protein). In some embodiments, the polypeptide is a mutant or a variant of any of the polypeptides described herein. In certain instances, the coding sequence for the polypeptides described herein are codon optimized for expression in a particular host cell. For example, for expression in E. coli, one or more codons can be optimized as described in, e.g., Grosjean et al., Gene 18:199-209 (1982).

The term “regulatory sequences” as used herein typically refers to a sequence of bases in DNA, operably-linked to DNA sequences encoding a protein that ultimately controls the expression of the protein. Examples of regulatory sequences include, but are not limited to, RNA promoter sequences, transcription factor binding sequences, transcription termination sequences, modulators of transcription (such as enhancer elements), nucleotide sequences that affect RNA stability, and translational regulatory sequences (such as, ribosome binding sites (e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences in eukaryotes), initiation codons, termination codons).

As used herein, the phrase “the expression of said nucleotide sequence is modified relative to the wild type nucleotide sequence,” means an increase or decrease in the level of expression and/or activity of an endogenous nucleotide sequence or the expression and/or activity of a heterologous or non-native polypeptide-encoding nucleotide sequence.

As used herein, the term “express” with respect to a polynucleotide is to cause it to function. A polynucleotide which encodes a polypeptide (or protein) will, when expressed, be transcribed and translated to produce that polypeptide (or protein). As used herein, the term “overexpress” means to express or cause to be expressed a polynucleotide or polypeptide in a cell at a greater concentration than is normally expressed in a corresponding wild-type cell under the same conditions.

The terms “altered level of expression” and “modified level of expression” are used interchangeably and mean that a polynucleotide, polypeptide, or hydrocarbon is present in a different concentration in an engineered host cell as compared to its concentration in a corresponding wild-type cell under the same conditions.

As used herein, the term “titer” refers to the quantity of fatty acid derivative produced per unit volume of host cell culture. In any aspect of the compositions and methods described herein, a fatty acid derivative is produced at a titer of about 25 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L, about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about 525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625 mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L, about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L, about 850 mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about 950 mg/L, about 975 mg/L, about 1000 mg/L, about 1050 mg/L, about 1075 mg/L, about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175 mg/L, about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275 mg/L, about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375 mg/L, about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475 mg/L, about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575 mg/L, about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675 mg/L, about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775 mg/L, about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875 mg/L, about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975 mg/L, about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or a range bounded by any two of the foregoing values. In other embodiments, a fatty acid derivative is produced at a titer of more than 100 g/L, more than 200 g/L, more than 300 g/L, or higher, such as 500 g/L, 700 g/L, 1000 g/L, 1200 g/L, 1500 g/L, or 2000 g/L. The preferred titer of fatty acid derivative produced by a recombinant host cell according to the methods of the invention is from 5 g/L to 200 g/L, 10 g/L to 150 g/L, 20 g/L to 120 g/L and 30 g/L to 100 g/L. The titer may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.

As used herein, the “yield of fatty acid derivative produced by a host cell” refers to the efficiency by which an input carbon source is converted to product (i.e., fatty alcohol or fatty aldehyde) in a host cell. Host cells engineered to produce fatty acid derivatives according to the methods of the invention have a yield of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% or a range bounded by any two of the foregoing values. In other embodiments, a fatty acid derivative or derivatives is produced at a yield of more than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Alternatively, or in addition, the yield is about 30% or less, about 27% or less, about 25% or less, or about 22% or less. Thus, the yield can be bounded by any two of the above endpoints. For example, the yield of a fatty acid derivative or derivatives produced by the recombinant host cell according to the methods of the invention can be 5% to 15%, 10% to 25%, 10% to 22%, 15% to 27%, 18% to 22%, 20% to 28%, or 20% to 30%. The yield may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.

As used herein, the term “productivity” refers to the quantity of a fatty acid derivative or derivatives produced per unit volume of host cell culture per unit time. In any aspect of the compositions and methods described herein, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell is at least 100 mg/L/hour, at least 200 mg/L/hour, at least 300 mg/L/hour, at least 400 mg/L/hour, at least 500 mg/L/hour, at least 600 mg/L/hour, at least 700 mg/L/hour, at least 800 mg/L/hour, at least 900 mg/L/hour, at least 1000 mg/L/hour, at least 1100 mg/L/hour, at least 1200 mg/L/hour, at least 1300 mg/L/hour, at least 1400 mg/L/hour, at least 1500 mg/L/hour, at least 1600 mg/L/hour, at least 1700 mg/L/hour, at least 1800 mg/L/hour, at least 1900 mg/L/hour, at least 2000 mg/L/hour, at least 2100 mg/L/hour, at least 2200 mg/L/hour, at least 2300 mg/L/hour, at least 2400 mg/L/hour, or at least 2500 mg/L/hour. For example, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell according to the methods of the may be from 500 mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to 2000 mg/L/hour. The productivity may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.

As used herein, the term “total fatty species” and “total fatty acid product” may be used interchangeably herein with reference to the amount of fatty alcohols, fatty aldehydes and fatty acids, as evaluated by GC-FID as described in International Patent Application Publication WO 2008/119082. The same terms may be used to mean fatty esters and free fatty acids when referring to a fatty ester analysis.

As used herein, the term “glucose utilization rate” means the amount of glucose used by the culture per unit time, reported as grams/liter/hour (g/L/hr).

As used herein, the term “carbon source” refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂). Exemplary carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acids, succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. The carbon source can also be a product of photosynthesis, such as glucose. In certain preferred embodiments, the carbon source is biomass. In other preferred embodiments, the carbon source is glucose. In other preferred embodiments the carbon source is sucrose.

As used herein, the term “biomass” refers to any biological material from which a carbon source is derived. In some embodiments, a biomass is processed into a carbon source, which is suitable for bioconversion. In other embodiments, the biomass does not require further processing into a carbon source. The carbon source can be converted into a biofuel. An exemplary source of biomass is plant matter or vegetation, such as corn, sugar cane, or switchgrass. Another exemplary source of biomass is metabolic waste products, such as animal matter (e.g., cow manure). Further exemplary sources of biomass include algae and other marine plants. Biomass also includes waste products from industry, agriculture, forestry, and households, including, but not limited to, fermentation waste, ensilage, straw, lumber, sewage, garbage, cellulosic urban waste, and food leftovers. The term “biomass” also can refer to sources of carbon, such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).

As used herein, the term “isolated,” with respect to products (such as fatty acids and derivatives thereof) refers to products that are separated from cellular components, cell culture media, or chemical or synthetic precursors. The fatty acids and derivatives thereof produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the fatty acids and derivatives thereof can collect in an organic phase either intracellularly or extracellularly.

As used herein, the terms “purify,” “purified,” or “purification” mean the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free (e.g., at least about 70% free, at least about 75% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of fatty acid derivatives in a sample. For example, when a fatty acid derivative is produced in a recombinant host cell, the fatty acid derivative can be purified by the removal of host cell proteins. After purification, the percentage of fatty acid derivative in the sample is increased. The terms “purify,” “purified,” and “purification” are relative terms which do not require absolute purity. Thus, for example, when a fatty acid derivative is produced in recombinant host cells, a purified fatty acid derivative is a fatty acid derivative that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).

General Overview of the Invention

In the compositions and methods of the invention, the production of a desired fatty acid derivative composition (e.g., acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones is enhanced by modifying the expression of one or more genes involved in a biosynthetic pathway for fatty acid, fatty ester, alkane, alkene, olefin, or fatty alcohol, production, degradation and/or secretion.

The invention provides recombinant host cells which have been engineered to provide enhanced fatty acid biosynthesis relative to non-engineered or native host cells (for example by strain improvements, as further described herein below).

The disclosure identifies polynucleotides useful in the recombinant host cells, methods, and compositions of the invention; however it will be recognized that absolute sequence identity to such polynucleotides is not necessary. For example, changes in a particular polynucleotide sequence can be made and the encoded polypeptide screened for activity. Such changes typically comprise conservative mutations and silent mutations (such as, for example, codon optimization). Modified or mutated (i.e., mutant) polynucleotides and encoded variant polypeptides can be screened for a desired function, such as, an improved function compared to the parent polypeptide, including but not limited to increased catalytic activity, increased stability, or decreased inhibition (e.g., decreased feedback inhibition), using methods known in the art. The disclosure identifies enzymatic activities involved in various steps (i.e., reactions) of the fatty acid biosynthetic pathways described herein according to Enzyme Classification (EC) number, and provides exemplary polypeptides (i.e., enzymes) categorized by such EC numbers, and exemplary polynucleotides encoding such polypeptides. Such exemplary polypeptides and polynucleotides, which are identified herein by Accession Numbers and/or Sequence Identifier Numbers (SEQ ID NOs), are useful for engineering fatty acid pathways in parental host cells to obtain the recombinant host cells described herein. It is to be understood, however, that polypeptides and polynucleotides described herein are exemplary and non-limiting. The sequences of homologues of exemplary polypeptides described herein are available to those of skill in the art using databases such as, for example, the Entrez databases provided by the National Center for Biotechnology Information (NCBI), the ExPasy databases provided by the Swiss Institute of Bioinformatics, the BRENDA database provided by the Technical University of Braunschweig, and the KEGG database provided by the Bioinformatics Center of Kyoto University and University of Tokyo, all which are available on the World Wide Web.

A variety of host cells can be modified to contain a fatty acid biosynthetic pathway such as those described herein, resulting in recombinant host cells suitable for the production of fatty acid derivatives. It is understood that a variety of cells can provide sources of genetic material, including polynucleotide sequences that encode polypeptides suitable for use in a recombinant host cell provided herein.

Strain Improvements

In order generate a high titer, yield, and productivity of fatty acid derivatives, a number of modifications were made to the production host cells.

FadR is a key regulatory factor involved in fatty acid degradation and fatty acid biosynthetic pathways (Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)). The E. coli ACS enzyme FadD and the fatty acid transport protein FadL are essential components of a fatty acid uptake system. FadL mediates transport of fatty acids into the bacterial cell, and FadD mediates formation of acyl-CoA esters. When no other carbon source is available, exogenous fatty acids are taken up by bacteria and converted to acyl-CoA esters, which can bind to the transcription factor FadR and derepress the expression of the fad genes that encode proteins responsible for fatty acid transport (FadL), activation (FadD), and β-oxidation (FadA, FadB, FadE, and FadH). When alternative sources of carbon are available, bacteria synthesize fatty acids as acyl-ACPs, which are used for phospholipid synthesis, but are not substrates for β-oxidation. Thus, acyl-CoA and acyl-ACP are both independent sources of fatty acids can result in different end-products (Caviglia et al., J. Biol. Chem., 279(12): 1163-1169 (2004)). U.S. Provisional Application No. 61/470,989 describes improved methods of producing fatty acid derivatives in a host cell which is genetically engineered to have an altered level of expression of a FadR polypeptide as compared to the level of expression of the FadR polypeptide in a corresponding wild-type host cell.

There are conflicting reports in the literature as to factors that can limit fatty acid biosynthesis in host cells, such as E. coli. One suggestion is that a limitation of the main precursors for fatty acid biosynthesis, for example, acetyl-CoA and malonyl-CoA can result in decreased synthesis of fatty acid derivatives. One approach to increasing the flux through fatty acid biosynthesis is to manipulate various enzymes in the pathway (FIGS. 1-2). Example 2 describes studies which show construction of fab operons that encode enzymes in the biosynthetic pathway for conversion of malonyl-CoA into acyl-ACPs and integration into the chromosome of an E. coli host cell as a means to increase the flux of fatty acid biosynthesis.

The supply of acyl-ACPs from acetyl-CoA via the acetyl-CoA carboxylase (acc) complex and fatty acid biosynthetic (fab) pathway is another step that may limit the rate of fatty acid derivative production (FIG. 3). In a study detailed in Example 3, the effect of overexpression of an optimized version of E. coli Corynebacterium glutamicum accABCD (±birA) demonstrated that such genetic modifications can lead to increased production of acetyl-coA and malonyl-CoA in E. coli.

In yet another approach, mutations in the rph and ilvG genes in the E. coli host cell were shown to result in higher free fatty acid (FFA) production, which translated into higher production of fatty alcohol. See Example 4.

In still another approach, transposon mutagenesis and high-throughput screening was carried out to find beneficial mutations that increase the titer or yield. Example 5 describes studies where it was observed that a transposon insertion in the yijP gene can improve the fatty alcohol yield in shake flask and fed-batch fermentations.

Generation of Fatty Acid Derivative by Recombinant Host Cells

This disclosure provides numerous examples of polypeptides (i.e., enzymes) having activities suitable for use in the fatty acid biosynthetic pathways described herein. Such polypeptides are collectively referred to herein as “fatty acid biosynthetic polypeptides” or “fatty acid biosynthetic enzymes”. Non-limiting examples of fatty acid pathway polypeptides suitable for use in recombinant host cells of the invention are provided herein.

In some embodiments, the invention includes a recombinant host cell comprising a polynucleotide sequence (also referred to herein as a “fatty acid biosynthetic polynucleotide” sequence) which encodes a fatty acid biosynthetic polypeptide.

The polynucleotide sequence, which comprises an open reading frame encoding a fatty acid biosynthetic polypeptide and operably-linked regulatory sequences, can be integrated into a chromosome of the recombinant host cells, incorporated in one or more plasmid expression systems resident in the recombinant host cell, or both. In the Examples, both plasmid expression systems and integration into the host genome are used to illustrate different embodiments of the present invention.

In some embodiments, a fatty acid biosynthetic polynucleotide sequence encodes a polypeptide which is endogenous to the parental host cell of the recombinant cell being engineered. Some such endogenous polypeptides are overexpressed in the recombinant host cell. An “endogenous polypeptide”, as used herein, refers to a polypeptide which is encoded by the genome of the parental (e.g., wild-type) cell that is engineered to produce the recombinant host cell.

In some embodiments, the fatty acid biosynthetic polynucleotide sequence encodes an exogenous or heterologous polypeptide. In other words, the polypeptide encoded by the polynucleotide is exogenous to the parental host cell. An “exogenous” (or “heterologous”) polypeptide, as used herein, refers to a polypeptide not encoded by the genome of the parental (e.g., wild-type) host cell that is being engineered to produce the recombinant host cell. Such a polypeptide can also be referred to as a “non-native” polypeptide. A variant (that is, a mutant) polypeptide is an example of a heterologous polypeptide.

In certain embodiments, the genetically modified host cell overexpresses a gene encoding a polypeptide (protein) that increases the rate at which the host cell produces the substrate of a fatty acid biosynthetic enzyme, i.e., a fatty acyl-thioester substrate. In certain embodiments, the enzyme encoded by the over expressed gene is directly involved in fatty acid biosynthesis.

Such recombinant host cells may be further engineered to comprise a polynucleotide sequence encoding one or more “fatty acid biosynthetic polypeptides”, (enzymes involved in fatty acid biosynthesis), for example, a polypeptide:

(1) having thioesterase activity, wherein the recombinant host cell synthesizes fatty acids;

(2) having thioesterase activity and carboxylic acid reductase (“CAR”) activity, wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols;

(3) having thioesterase activity, carboxylic acid reductase activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols;

(4) having acyl-CoA reductase (“AAR”) activity wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols;

(5) having acyl-CoA reductase (“AAR”) activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols;

(6) having fatty alcohol forming acyl-CoA reductase (“FAR”) activity, wherein the recombinant host cell synthesizes fatty alcohols;

(7) having thioesterase activity, carboxylic acid reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes;

(8) having acyl-CoA reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes

(9) having ester synthase activity wherein the recombinant host cell synthesizes fatty esters (“one enzyme system”; FIG. 5);

(10) having thioesterase activity, acyl-CoA synthase activity and ester synthase activity wherein the recombinant host cell synthesizes fatty esters (“three enzyme system”; FIG. 5);

(11) having OleA activity, wherein the recombinant host cell synthesizes aliphatic ketones;

(12) having OleABCD activity, wherein the recombinant host cell synthesizes internal olefins, or

(13) having thioesterase activity and decarboxylase activity, wherein the recombinant host cell synthesizes terminal olefins.

In some embodiments, at least one polypeptide encoded by a fatty acid biosynthetic polynucleotide is an exogenous (or heterologous) polypeptide (for example, a polypeptide originating from an organism other than the parental host cell, or, a variant of a polypeptide native to the parental microbial cell) or an endogenous polypeptide (that is, a polypeptide native to the parental host cell) wherein the endogenous polypeptide is overexpressed in the recombinant host cell.

Table 1 provides a listing of exemplary proteins which can be expressed in recombinant host cells to facilitate production of particular fatty acid derivatives.

TABLE 1 Gene Designations Gene EC Exemplary Designation Source Organism Enzyme Name Accession No. Number Use 1. Fatty Acid Production Increase/Product Production Increase accA E. coli, Lactococci Acetyl-CoA AAC73296, 6.4.1.2 increase carboxylase, subunit A NP_414727 Malonyl-CoA (carboxyltransferase production alpha) accB E. coli, Lactococci Acetyl-CoA NP_417721 6.4.1.2 increase carboxylase, subunit B Malonyl-CoA (BCCP: biotin production carboxyl carrier protein) accC E. coli, Lactococci Acetyl-CoA NP_417722 6.4.1.2, increase carboxylase, subunit C 6.3.4.14 Malonyl-CoA (biotin carboxylase) production accD E. coli, Lactococci Acetyl-CoA NP_416819 6.4.1.2 increase carboxylase, subunit D Malonyl-CoA (carboxyltransferase production beta) fadD E. coli W3110 acyl-CoA synthase AP_002424 2.3.1.86, increase Fatty 6.2.1.3 acid production fabA E. coli K12 β-hydroxydecanoyl NP_415474 4.2.1.60 increase fatty thioester acyl- dehydratase/isomerase ACP/CoA production fabB E. coli 3-oxoacyl-[acyl- BAA16180 2.3.1.41 increase fatty carrier-protein] acyl- synthase I ACP/CoA production fabD E. coli K12 [acyl-carrier-protein] AAC74176 2.3.1.39 increase fatty S-malonyltransferase acyl- ACP/CoA production fabF E. coli K12 3-oxoacyl-[acyl- AAC74179 2.3.1.179 increase fatty carrier-protein] acyl- synthase II ACP/CoA production fabG E. coli K12 3-oxoacyl-[acyl-carrier AAC74177 1.1.1.100 increase fatty protein] reductase acyl- ACP/CoA production fabH E. coli K12 3-oxoacyl-[acyl- AAC74175 2.3.1.180 increase fatty carrier-protein] acyl- synthase III ACP/CoA production fabI E. coli K12 enoyl-[acyl-carrier- NP_415804 1.3.1.9 increase fatty protein] reductase acyl- ACP/CoA production fabR E. coli K12 Transcriptional NP_418398 none modulate Repressor unsaturated fatty acid production fabV Vibrio cholerae enoyl-[acyl-carrier- YP_001217283 1.3.1.9 increase fatty protein] reductase acyl- ACP/CoA production fabZ E. coli K12 (3R)-hydroxymyristol NP_414722 4.2.1.— increase fatty acyl carrier protein acyl- dehydratase ACP/CoA production fadE E. coli K13 acyl-CoA AAC73325 1.3.99.3, reduce fatty dehydrogenase 1.3.99.— acid degradation fadR E. coli transcriptional NP_415705 none Block or regulatory protein reverse fatty acid degradation 2. Chain Length Control tesA (with or E. coli thioesterase - leader P0ADA1 3.1.2.—, C18 Chain without leader sequence is amino 3.1.1.5 Length sequence) acids 1-26 tesA (without E. coli thioesterase AAC73596, 3.1.2.—, C18:1 Chain leader NP_415027 3.1.1.5 Length sequence) tesA (mutant E. coli thioesterase L109P 3.1.2.—, <C18 Chain of E. coli 3.1.1.5 Length thioesterase I complexed with octanoic acid) fatB1 Umbellularia thioesterase Q41635 3.1.2.14 C12:0 Chain californica Length fatB2 Cuphea hookeriana thioesterase AAC49269 3.1.2.14 C8:0-C10:0 Chain Length fatB3 Cuphea hookeriana thioesterase AAC72881 3.1.2.14 C14:0-C16:0 Chain Length fatB Cinnamomumcamphora thioesterase Q39473 3.1.2.14 C14:0 Chain Length fatB Arabidopsis thioesterase CAA85388 3.1.2.14 C16:1 Chain thaliana Length fatA1 Helianthus annuus thioesterase AAL79361 3.1.2.14 C18:1 Chain Length atfata Arabidopsis thioesterase NP_189147, 3.1.2.14 C18:1 Chain thaliana NP_193041 Length fatA Brassica juncea thioesterase CAC39106 3.1.2.14 C18:1 Chain Length fatA Cuphea hookeriana thioesterase AAC72883 3.1.2.14 C18:1 Chain Length tes Photbacterium thioesterase YP_130990 3.1.2.14 Chain Length profundum tesB E. coli thioesterase NP_414986 3.1.2.14 Chain Length fadM E. coli thioesterase NP_414977 3.1.2.14 Chain Length yciA E. coli thioesterase NP_415769 3.1.2.14 Chain Length ybgC E. coli thioesterase NP_415264 3.1.2.14 Chain Length 3. Saturation Level Control* Sfa E. coli Suppressor of fabA AAN79592, none increase AAC44390 monounsaturated fatty acids fabA E. coli K12 β-hydroxydecanoyl NP_415474 4.2.1.60 produce thioester unsaturated dehydratase/isomerase fatty acids GnsA E. coli suppressors of the ABD18647.1 none increase secG null mutation unsaturated fatty acid esters GnsB E. coli suppressors of the AAC74076.1 none increase secG null mutation unsaturated fatty acid esters fabB E. coli 3-oxoacyl-[acyl- BAA16180 2.3.1.41 modulate carrier-protein] unsaturated synthase I fatty acid production des Bacillus subtilis D5 fatty acyl O34653 1.14.19 modulate desaturase unsaturated fatty acid production 4. Product Output: wax production AT3G51970 Arabidopsis long-chain-alcohol O- NP_190765 2.3.1.26 wax thaliana fatty-acyltransferase production ELO1 Pichia angusta Fatty acid elongase BAD98251 2.3.1.— produce very long chain length fatty acids plsC Saccharomyces acyltransferase AAA16514 2.3.1.51 wax cerevisiae production DAGAT/DGAT Arabidopsis diacylglycerol AAF19262 2.3.1.20 wax thaliana acyltransferase production hWS Homo sapiens acyl-CoA wax alcohol AAX48018 2.3.1.20 wax acyltransferase production aft1 Acinetobacter sp. bifunctional wax ester AAO17391 2.3.1.20 wax ADP1 synthase/acyl- production CoA:diacylglycerol acyltransferase ES9 Marinobacter wax ester synthase ABO21021 2.3.1.20 wax hydrocarbonoclasticus production mWS Simmondsia wax ester synthase AAD38041 2.3.1.— wax chinensis production 5. Product Output: Fatty Alcohol Output thioesterases (see increase fatty above) acid/fatty alcohol production BmFAR Bombyxmori FAR (fatty alcohol BAC79425 1.1.1.— convert acyl- forming acyl-CoA CoA to fatty reductase) alcohol acr1 Acinetobacter sp. acyl-CoA reductase YP_047869 1.2.1.42 reduce fatty ADP1 acyl-CoA to fatty aldehydes yqhD E. coli W3110 alcohol dehydrogenase AP_003562 1.1.—.— reduce fatty aldehydes to fatty alcohols; increase fatty alcohol production alrA Acinetobacter sp. alcohol dehydrogenase CAG70252 1.1.—.— reduce fatty ADP1 aldehydes to fatty alcohols BmFAR Bombyxmori FAR (fatty alcohol BAC79425 1.1.1.— reduce fatty forming acyl-CoA acyl-CoA to reductase) fatty alcohol GTNG_1865 Geobacillusthermod Long-chain aldehyde YP_001125970 1.2.1.3 reduce fatty enitrificans NG80-2 dehydrogenase aldehydes to fatty alcohols AAR Synechococcus Acyl-ACP reductase YP_400611 1.2.1.42 reduce fatty elongatus acyl- ACP/CoA to fatty aldehydes carB Mycobacterium carboxylic acid YP_889972 6.2.1.3, reduce fatty smegmatis reductase protein 1.2.1.42 acids to fatty aldehyde FadD E. coli K12 acyl-CoA synthetase NP_416319 6.2.1.3 activates fatty acids to fatty acyl-CoAs atoB Erwinia carotovora acetyl-CoA YP_049388 2.3.1.9 production of acetyltransferase butanol hbd Butyrivibrio fibrisolvens Beta-hydroxybutyryl- BAD51424 1.1.1.157 production of CoA dehydrogenase butanol CPE0095 Clostridium crotonasebutyryl-CoA BAB79801 4.2.1.55 production of perfringens dehydryogenase butanol bcd Clostridium butyryl-CoA AAM14583 1.3.99.2 production of beijerinckii dehydryogenase butanol ALDH Clostridium coenzyme A-acylating AAT66436 1.2.1.3 production of beijerinckii aldehyde butanol dehydrogenase AdhE E. coli CFT073 aldehyde-alcohol AAN80172 1.1.1.1 production of dehydrogenase 1.2.1.10 butanol 6. Fatty Alcohol Acetyl Ester Output thioesterases (see modify output above) acr1 Acinetobacter sp. acyl-CoA reductase YP_047869 1.2.1.42 modify output ADP1 yqhD E. Coli K12 alcohol dehydrogenase AP_003562 1.1.—.— modify output AAT Fragaria x alcohol O- AAG13130 2.3.1.84 modify output ananassa acetyltransferase 7. Product Export AtMRP5 Arabidopsis Arabidopsis thaliana NP_171908 none modify thaliana multidrug resistance- product associated export amount AmiS2 Rhodococcus sp. ABC transporter JC5491 none modify AmiS2 product export amount AtPGP1 Arabidopsis Arabidopsis thaliana p NP_181228 none modify thaliana glycoprotein 1 product export amount AcrA Candidatus Protochlamydia amoebophila putative multidrug- CAF23274 none modify UWE25 efflux transport protein product acrA export amount AcrB Candidatus Protochlamydia amoebophila probable multidrug- CAF23275 none modify UWE25 efflux transport product protein, acrB export amount TolC Francisella tularensis Outer membrane ABD59001 none modify subsp. novicida protein [Cell envelope product biogenesis, export amount AcrE Shigella sonnei transmembrane protein YP_312213 none modify Ss046 affects septum product formation and cell export membrane amount permeability AcrF E. coli Acriflavine resistance P24181 none modify protein F product export amount tll1619 Thermosynechococcus multidrug efflux NP_682409.1 none modify elongatus [BP-1] transporter product export amount tll0139 Thermosynechococcus multidrug efflux NP_680930.1 none modify elongatus [BP-1] transporter product export amount 8. Fermentation replication increase checkpoint output genes efficiency umuD Shigella sonnei DNA polymerase V, YP_310132 3.4.21.— increase Ss046 subunit output efficiency umuC E. coli DNA polymerase V, ABC42261 2.7.7.7 increase subunit output efficiency pntA, pntB Shigella flexneri NADH:NADPH P07001, 1.6.1.2 increase transhydrogenase P0AB70 output (alpha and beta efficiency subunits) 9. Other fabK Streptococcus trans-2-enoyl-ACP AAF98273 1.3.1.9 Contributes to pneumoniae reductase II fatty acid biosynthesis fabL Bacillus enoyl-(acyl carrier AAU39821 1.3.1.9 Contributes to licheniformis DSM protein) reductase fatty acid 13 biosynthesis fabM Streptococcus trans-2,cis-3- DAA05501 4.2.1.17 Contributes to mutans decenoyl-ACP fatty acid isomerase biosynthesis

Production of Fatty Acids

The recombinant host cells may comprise one or more polynucleotide sequences that comprise an open reading frame encoding a thioesterase, e.g., having an Enzyme Commission number of EC 3.1.1.5 or EC 3.1.2.—(for example, EC 3.1.2.14), together with operably-linked regulatory sequences that facilitate expression of the protein in the recombinant host cells. In the recombinant host cells, the open reading frame coding sequences and/or the regulatory sequences are modified relative to the corresponding wild-type gene encoding the thioesterase. The activity of the thioesterase in the recombinant host cell is modified relative to the activity of the thioesterase expressed from the corresponding wild-type gene in a corresponding host cell. In some embodiments, a fatty acid derivative composition comprising fatty acids is produced by culturing a recombinant cell in the presence of a carbon source under conditions effective to express the thioesterase.

In related embodiments, the recombinant host cell comprises a polynucleotide encoding a polypeptide having thioesterase activity, and one or more additional polynucleotides encoding polypeptides having other fatty acid biosynthetic enzyme activities. In some such instances, the fatty acid produced by the action of the thioesterase is converted by one or more enzymes having a different fatty acid biosynthetic enzyme activity to another fatty acid derivative, such as, for example, a fatty ester, fatty aldehyde, fatty alcohol, or a hydrocarbon.

The chain length of a fatty acid, or a fatty acid derivative made therefrom, can be selected for by modifying the expression of particular thioesterases. The thioesterase will influence the chain length of fatty acid derivatives produced. The chain length of a fatty acid derivative substrate can be selected for by modifying the expression of selected thioesterases (EC 3.1.2.14 or LC 3, 1.1.5). Hence, host cells can be engineered to express, overexpress, have attenuated expression, or not express one or more selected thioesterases to increase the production of a preferred fatty acid derivative substrate. For example, C₁₀ fatty acids can be produced by expressing a thioesterase that has a preference for producing C₁₀ fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than C₁₀ fatty acids (e.g., a thioesterase which prefers to produce C₁₄ fatty acids). This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 10. In other instances, C₁₄ fatty acids can be produced by attenuating endogenous thioesterases that produce non-C₁₄ fatty acids and expressing the thioesterases that use C₁₄-ACP. In some situations, C₁₂ fatty acids can be produced by expressing thioesterases that use C₁₂-ACP and attenuating thioesterases that produce non-C₁₂ fatty acids. For example, C12 fatty acids can be produced by expressing a thioesterase that has a preference for producing C12 fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than C12 fatty acids. This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 12. The fatty acid derivatives are recovered from the culture medium with substantially all of the fatty acid derivatives produced extracellularly. The fatty acid derivative composition produced by a recombinant host cell can be analyzed using methods known in the art, for example, GC-FID, in order to determine the distribution of particular fatty acid derivatives as well as chain lengths and degree of saturation of the components of the fatty acid derivative composition. Acetyl-CoA, malonyl-CoA, and fatty acid overproduction can be verified using methods known in the art, for example, by using radioactive precursors, HPLC, or GC-MS subsequent to cell lysis.

Additional non-limiting examples of thioesterases and polynucleotides encoding them for use in the fatty acid pathway are provided in PCT Publication No. WO 2010/075483, expressly incorporated by reference herein.

Production of Fatty Aldehydes

In one embodiment, the recombinant host cell produces a fatty aldehyde. In some embodiments, a fatty acid produced by the recombinant host cell is converted into a fatty aldehyde. In some embodiments, the fatty aldehyde produced by the recombinant host cell is then converted into a fatty alcohol or a hydrocarbon.

In some embodiments, native (endogenous) fatty aldehyde biosynthetic polypeptides, such as aldehyde reductases, are present in the host cell (e.g., E. coli) and are effective to convert fatty aldehydes to fatty alcohols. In other embodiments, a native (endogenous) fatty aldehyde biosynthetic polypeptide is overexpressed. In still other embodiments, an exogenous fatty aldehyde biosynthetic polypeptide is introduced into a recombinant host cell and expressed or overexpressed.

A native or recombinant host cell may comprise a polynucleotide encoding an enzyme having fatty aldehyde biosynthesis activity (also referred to herein as a “fatty aldehyde biosynthetic polypeptide” or a “fatty aldehyde biosynthetic polypeptide” or enzyme). A fatty aldehyde is produced when the fatty aldehyde biosynthetic enzyme is expressed or overexpressed in the host cell.

A recombinant host cell engineered to produce a fatty aldehyde will typically convert some of the fatty aldehyde to a fatty alcohol.

In some embodiments, a fatty aldehyde is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having fatty aldehyde biosynthetic activity such as carboxylic acid reductase (CAR) activity.

The terms “carboxylic acid reductase,” and “CAR,” are used interchangeably herein with respect to a “fatty aldehyde biosynthetic polypeptide”. CarB, is an exemplary carboxylic acid reductase. In practicing the invention, a gene encoding a carboxylic acid reductase polypeptide may be expressed or overexpressed in the host cell. In some embodiments, the CarB polypeptide has the amino acid sequence of SEQ ID NO: 7. In other embodiments, the CarB polypeptide is a variant or mutant of SEQ ID NO: 7.

Examples of carboxylic acid reductase (CAR) polypeptides and polynucleotides encoding them include, but are not limited to FadD9 (EC 6.2.1.-, UniProtKB Q50631, GenBank NP_(—)217106, SEQ ID NO: 34), CarA (GenBank ABK75684), CarB (GenBank YP889972; SEQ ID NO: 33) and related polypeptides described in PCT Publication No. WO 2010/042664 and U.S. Pat. No. 8,097,439, each of which is expressly incorporated by reference herein. In some embodiments the recombinant host cell further comprises a polynucleotide encoding a thioesterase.

In some embodiments, the fatty aldehyde is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a fatty aldehyde biosynthetic polypeptide, such as a polypeptide having acyl-ACP reductase (AAR) activity. Expression of acyl-ACP reductase in a recombinant host cell results in the production of fatty aldehydes and fatty alcohols. (See FIG. 4.) Native (endogenous) aldehyde reductases present in a recombinant host cell (e.g., E. coli), can convert fatty aldehydes into fatty alcohols. Exemplary acyl-ACP reductase polypeptides are described in PCT Publication Nos. WO2009/140695 and WO/2009/140696, both of which are expressly incorporated by reference herein.

A composition comprising fatty aldehydes (“a fatty aldehyde composition”) is produced by culturing a host cell in the presence of a carbon source under conditions effective to express the fatty aldehyde biosynthetic enzyme. In some embodiments, the fatty aldehyde composition comprises fatty aldehydes and fatty alcohols. Typically, the fatty aldehyde composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium.

Production of Fatty Alcohols

In some embodiments, the recombinant host cell comprises a polynucleotide encoding a polypeptide (an enzyme) having fatty alcohol biosynthetic activity (also referred to herein as a “fatty alcohol biosynthetic polypeptide” or a “fatty alcohol biosynthetic enzyme”), and a fatty alcohol is produced by the recombinant host cell. A composition comprising fatty alcohols (“a fatty alcohol composition”) may be produced by culturing the recombinant host cell in the presence of a carbon source under conditions effective to express a fatty alcohol biosynthetic enzyme. In some embodiments, the fatty alcohol composition comprises fatty alcohols, however, a fatty alcohol composition may comprise other fatty acid derivatives. Typically, the fatty alcohol composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium.

In one approach, recombinant host cells have been engineered to produce fatty alcohols by expressing a thioesterase, which catalyzes the conversion of acyl-ACPs into free fatty acids (FFAs) and a carboxylic acid reductase (CAR), which converts free fatty acids into fatty aldehydes. Native (endogenous) aldehyde reductases present in the host cell (e.g., E. coli) can convert the fatty aldehydes into fatty alcohols.

In some embodiments, native (endogenous) fatty aldehyde biosynthetic polypeptides, such as aldehyde reductases present in the host cell, may be sufficient to convert fatty aldehydes to fatty alcohols. However, in other embodiments, a native (endogenous) fatty aldehyde biosynthetic polypeptide is overexpressed and in still other embodiments, an exogenous fatty aldehyde biosynthetic polypeptide is introduced into a recombinant host cell and expressed or overexpressed.

In some embodiments, the fatty alcohol is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having fatty alcohol biosynthetic activity which converts a fatty aldehyde to a fatty alcohol. For example, an “alcohol dehydrogenase” (also referred to herein as an “aldehyde reductase”, e.g., EC 1.1.1.1), may be used in practicing the invention. As used herein, the term “alcohol dehydrogenase” refers to a polypeptide capable of catalyzing the conversion of a fatty aldehyde to an alcohol (e.g., a fatty alcohol). One of ordinary skill in the art will appreciate that certain alcohol dehydrogenases are capable of catalyzing other reactions as well, and these non-specific alcohol dehydrogenases also are encompassed by the term “alcohol dehydrogenase.” Examples of alcohol dehydrogenase polypeptides useful in accordance with the invention include, but are not limited to A1rA of Acinetobacter sp. M-1 (SEQ ID NO: 3) or A1rA homologs such as AlrAadpl (SEQ ID NO: 4) and endogenous E. coli alcohol dehydrogenases such as YjgB, (AAC77226) (SEQ ID NO: 5), DkgA (NP_(—)417485), DkgB (NP_(—)414743), YdjL (AAC74846), YdjJ (NP_(—)416288), AdhP (NP_(—)415995), YhdH (NP_(—)417719), YahK (NP_(—)414859), YphC (AAC75598), YqhD (446856) and YbbO [AAC73595.1]. Additional examples are described in International Patent Application Publication Nos. WO 2007/136762, WO2008/119082 and WO 2010/062480, each of which is expressly incorporated by reference herein. In certain embodiments, the fatty alcohol biosynthetic polypeptide has aldehyde reductase or alcohol dehydrogenase activity (EC 1.1.1.1).

In another approach, recombinant host cells have been engineered to produce fatty alcohols by expressing fatty alcohol forming acyl-CoA reductases or fatty acyl reductases (“FARs”) which convert fatty acyl-thioester substrates (e.g., fatty acyl-CoA or fatty acyl-ACP) to fatty alcohols. In some embodiments, the fatty alcohol is produced by expressing or overexpressing a polynucleotide encoding a polypeptide having fatty alcohol forming acyl-CoA reductase (FAR) activity in a recombinant host cell. Examples of FAR polypeptides useful in accordance with this embodiment are described in PCT Publication No. WO 2010/062480 which is expressly incorporated by reference herein.

Fatty alcohol may be produced via an acyl-CoA dependent pathway utilizing fatty acyl-ACP and fatty acyl-CoA intermediates and an acyl-CoA independent pathway utilizing fatty acyl-ACP intermediates but not a fatty acyl-CoA intermediate. In particular embodiments, the enzyme encoded by the over expressed gene is selected from a fatty acid synthase, an acyl-ACP thioesterase, a fatty acyl-CoA synthase and an acetyl-CoA carboxylase. In some embodiments, the protein encoded by the over expressed gene is endogenous to the host cell. In other embodiments, the protein encoded by the overexpressed gene is heterologous to the host cell.

Fatty alcohols are also made in nature by enzymes that are able to reduce various acyl-ACP or acyl-CoA molecules to the corresponding primary alcohols. See also, U.S. Patent Publication Nos. 20100105963, and 20110206630 and U.S. Pat. No. 8,097,439, expressly incorporated by reference herein.

Strategies to increase production of fatty alcohols by recombinant host cells include increased flux through the fatty acid biosynthetic pathway by overexpression of native fatty acid biosynthetic genes and/or expression of exogenous fatty acid biosynthetic genes from different organisms in the production host such that fatty alcohol biosynthesis is increased.

Production of Esters

As used herein, the term “fatty ester” may be used with reference to an ester. A fatty ester as referred to herein can be any ester made from a fatty acid, for example a fatty acid ester. In some embodiments, a fatty ester contains an A side and a B side. As used herein, an “A side” of an ester refers to the carbon chain attached to the carboxylate oxygen of the ester. As used herein, a “B side” of an ester refers to the carbon chain comprising the parent carboxylate of the ester. In embodiments where the fatty ester is derived from the fatty acid biosynthetic pathway, the A side is contributed by an alcohol, and the B side is contributed by a fatty acid.

Any alcohol can be used to form the A side of the fatty esters. For example, the alcohol can be derived from the fatty acid biosynthetic pathway. Alternatively, the alcohol can be produced through non-fatty acid biosynthetic pathways. Moreover, the alcohol can be provided exogenously. For example, the alcohol can be supplied in the fermentation broth in instances where the fatty ester is produced by an organism. Alternatively, a carboxylic acid, such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol.

The carbon chains comprising the A side or B side can be of any length. In one embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. When the fatty ester is a fatty acid methyl ester, the A side of the ester is 1 carbon in length. When the fatty ester is a fatty acid ethyl ester, the A side of the ester is 2 carbons in length. The B side of the ester can be at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. The A side and/or the B side can be straight or branched chain. The branched chains can have one or more points of branching. In addition, the branched chains can include cyclic branches. Furthermore, the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.

In one embodiment, the fatty ester is produced biosynthetically. In this embodiment, first the fatty acid is “activated.” Non-limiting examples of “activated” fatty acids are acyl-CoA, acyl ACP, and acyl phosphate. Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation. In addition, acyl-CoA can be synthesized from a free fatty acid, a CoA, and an adenosine nucleotide triphosphate (ATP). An example of an enzyme which produces acyl-CoA is acyl-CoA synthase.

In some embodiments, the recombinant host cell comprises a polynucleotide encoding a polypeptide, e.g., an enzyme having ester synthase activity, (also referred to herein as an “ester synthase polypeptide” or an “ester synthase”). A fatty ester is produced by a reaction catalyzed by the ester synthase polypeptide expressed or overexpressed in the recombinant host cell. In some embodiments, a composition comprising fatty esters (also referred to herein as a “fatty ester composition”) comprising fatty esters is produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express an ester synthase. In some embodiments, the fatty ester composition is recovered from the cell culture.

Ester synthase polypeptides include, for example, an ester synthase polypeptide classified as EC 2.3.1.75, or any other polypeptide which catalyzes the conversion of an acyl-thioester to a fatty ester, including, without limitation, a thioesterase, an ester synthase, an acyl-CoA:alcohol transacylase, an acyltransferase, or a fatty acyl-CoA:fatty alcohol acyltransferase. For example, the polynucleotide may encode wax/dgat, a bifunctional ester synthase/acyl-CoA:diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobacter sp. Strain ADP, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, or Alkaligenes eutrophus. In a particular embodiment, the ester synthase polypeptide is an Acinetobacter sp. diacylglycerol O-acyltransferase (wax-dgaT; UniProtKB Q8GGG1, GenBank AA017391) or Simmondsia chinensis wax synthase (UniProtKB Q9XGY6, GenBank AAD38041. In another embodiment, the ester synthase polypeptide is for example ES9 (a wax ester synthase from Marinobacter hydrocarbonoclasticus DSM 8798, UniProtKB A3RE51 (SEQ ID NO: 6); ES8 of Marinobacter hydrocarbonoclasticus DSM8789 (GenBank Accession No. AB021021; SEQ ID NO:7); GenBank AB021021, encoded by the ws2 gene; or ES376 (another wax ester synthase derived from Marinobacter hydrocarbonoclasticus DSM 8798, UniProtKB A3RE50, GenBank AB021020, encoded by the ws1 gene. In a particular embodiment, the polynucleotide encoding the ester synthase polypeptide is overexpressed in the recombinant host cell.

In some embodiments, a fatty acid ester is produced by a recombinant host cell engineered to express three fatty acid biosynthetic enzymes: a thioesterase enzyme, an acyl-CoA synthetase (fadD) enzyme and an ester synthase enzyme (“three enzyme system”; FIG. 5).

In other embodiments, a fatty acid ester is produced by a recombinant host cell engineered to express one fatty acid biosynthetic enzyme, an ester synthase enzyme (“one enzyme system”; FIG. 5).

Non-limiting examples of ester synthase polypeptides and polynucleotides encoding them suitable for use in these embodiments include those described in PCT Publication Nos. WO 2007/136762 and WO2008/119082, and WO/2011/038134 (“three enzyme system”) and WO/2011/038132 (“one enzyme system”), each of which is expressly incorporated by reference herein.

The recombinant host cell may produce a fatty ester, such as a fatty acid methyl ester, a fatty acid ethyl ester or a wax ester in the extracellular environment of the host cells.

Production of Hydrocarbons

This aspect of the invention is based, at least in part, on the discovery that altering the level of expression of a fatty aldehyde biosynthetic polypeptide, for example, an acyl-ACP reductase polypeptide (EC 6.4.1.2) and a hydrocarbon biosynthetic polypeptide, e.g., a decarbonylase in a recombinant host cell facilitates enhanced production of hydrocarbons by the recombinant host cell.

In one embodiment, the recombinant host cell produces a hydrocarbon, such as an alkane or an alkene (e.g., a terminal olefin or an internal olefin) or a ketone. In some embodiments, a fatty aldehyde produced by a recombinant host cell is converted by decarboxylation, removing a carbon atom to form a hydrocarbon. In other embodiments, a fatty acid produced by a recombinant host cell is converted by decarboxylation, removing a carbon atom to form a terminal olefin. In some embodiments, an acyl-ACP intermediate is converted by decarboxylation, removing a carbon atom to form an internal olefin or a ketone. See, FIG. 6.

In some embodiments, the recombinant host cell comprises a polynucleotide encoding a polypeptide (an enzyme) having hydrocarbon biosynthetic activity (also referred to herein as a “hydrocarbon biosynthetic polypeptide” or a “hydrocarbon biosynthetic enzyme”), and the hydrocarbon is produced by expression or overexpression of the hydrocarbon biosynthetic enzyme in a recombinant host cell.

An alkane biosynthetic pathway from cyanobacteria consisting of an acyl-acyl carrier protein reductase (AAR) and an aldehyde decarbonylase (ADC), which together convert intermediates of fatty acid metabolism to alkanes and alkenes has been used to engineer recombinant host cells for the production of hydrocarbons (FIG. 6). The second of two reactions in the pathway through which saturated acyl-ACPs are converted to alkanes in cyanobacteria entails scission of the C1-C2 bond of a fatty aldehyde intermediate by the enzyme aldehyde decarbonylase (ADC), a ferritin-like protein with a binuclear metal cofactor of unknown composition.

In some embodiments, the hydrocarbon is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having hydrocarbon biosynthetic activity such as an aldehyde decarbonylase (ADC) activity (e.g., EC 4.1.99.5). exemplary polynucleotides encoding an aldehyde decarbonylase useful in accordance with this embodiment include, but are not limited to, those described in PCT Publication Nos. WO 2008/119082 and WO 2009/140695 which are expressly incorporated by reference herein and those sequences presented in Table 2, below. In some embodiments the recombinant host cell further comprises a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide. In some embodiments the recombinant host cell further comprises a polynucleotide encoding an acyl-ACP reductase. See, for example Table 2, below.

TABLE 2 Exemplary Hydrocarbon Biosynthetic Polynucleotides and Polypeptides. Polypeptide Nucleotide Protein name sequence sequence Sequence Decarbonylase SEQ ID SEQ ID NO: Synechococcus elongatus (ADC) NO: 35 36 PCC7942 YP.sub.--400610 (Synpcc7942.sub.--1593) Acyl-ACP SEQ ID SEQ ID NO: Synechococcus elongatus Reducatase NO: 37 38 PCC7942 YP_400611 (AAR) (Synpcc7942_1594) Decarbonylase SEQ ID SEQ ID NO: Prochlorococcus mariunus (ADC) NO: 39 40 CCMP1986 PMM0532 Acyl-ACP SEQ ID SEQ ID NO: Prochlorococcus marinus Reducatase NO: 41 42 CCMP1986 PMM0533 (AAR) (NP_892651)

In some embodiments, a composition comprising hydrocarbons (also referred to herein as a “hydrocarbon composition”) is produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the Acyl-CoA reductase and decarbonylase polynucleotides. In some embodiments, the hydrocarbon composition comprises saturated and unsaturated hydrocarbons, however, a hydrocarbon composition may comprise other fatty acid derivatives. Typically, the hydrocarbon composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium.

As used herein, the term “alkane” means saturated hydrocarbons or compounds that consist only of carbon (C) and hydrogen (H), wherein these atoms are linked together by single bonds (i.e., they are saturated compounds).

The terms “olefin” and “alkene” are used interchangeably herein, and refer to hydrocarbons containing at least one carbon-to-carbon double bond (i.e., they are unsaturated compounds).

The terms “terminal olefin,” “α-olefin”, “terminal alkene” and “1-alkene” are used interchangeably herein with reference to α-olefins or alkenes with a chemical formula CxH2x, distinguished from other olefins with a similar molecular formula by linearity of the hydrocarbon chain and the position of the double bond at the primary or alpha position.

In some embodiments, a terminal olefin is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having decarboxylase activity as described, for example, in PCT Publication No. WO 2009/085278 which is expressly incorporated by reference herein. In some embodiments the recombinant host cell further comprises a polynucleotide encoding a thioesterase.

In other embodiments, a ketone is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having OleA activity as described, for example, in PCT Publication No. WO 2008/147781, which is expressly incorporated by reference herein.

In related embodiments, an internal olefin is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having OleCD or OleBCD activity together with a polypeptide having OleA activity as described, for example, in PCT Publication No. WO 2008/147781, expressly incorporated by reference herein.

Recombinant Host Cells and Cell Cultures

Strategies to increase production of fatty acid derivatives by recombinant host cells include increased flux through the fatty acid biosynthetic pathway by overexpression of native fatty acid biosynthetic genes and expression of exogenous fatty acid biosynthetic genes from different organisms in the production host.

As used herein, the term “recombinant host cell” or “engineered host cell” refers to a host cell whose genetic makeup has been altered relative to the corresponding wild-type host cell, for example, by deliberate introduction of new genetic elements and/or deliberate modification of genetic elements naturally present in the host cell. The offspring of such recombinant host cells also contain these new and/or modified genetic elements. In any of the aspects of the invention described herein, the host cell can be selected from the group consisting of a plant cell, insect cell, fungus cell (e.g., a filamentous fungus, such as Candida sp., or a budding yeast, such as Saccharomyces sp.), an algal cell and a bacterial cell. In one preferred embodiment, recombinant host cells are “recombinant microorganisms.”

Examples of host cells that are microorganisms, include but are not limited to cells from the genus Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces. In some embodiments, the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell.

In some embodiments, the host cell is an E. coli cell.

In other embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

In other embodiments, the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell.

In yet other embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell.

In yet other embodiments, the host cell is an Actinomycetes cell.

In some embodiments, the host cell is a Saccharomyces cerevisiae cell.

In other embodiments, the host cell is a cell from a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, an engineered organism thereof, or a synthetic organism. In some embodiments, the host cell is light-dependent or fixes carbon. In some embodiments, the host cell has autotrophic activity. In some embodiments, the host cell has photoautotrophic activity, such as in the presence of light. In some embodiments, the host cell is heterotrophic or mixotrophic in the absence of light. In certain embodiments, the host cell is a cell from Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela sauna, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.

Production of Fatty Acid Derivative Compositions by Recombinant Host Cells

A large variety of fatty acid derivatives can be produced by recombinant host cells comprising strain improvements as described herein, including, but not limited to, fatty acids, acyl-CoA, fatty aldehydes, short and long chain alcohols, hydrocarbons (e.g., alkanes, alkenes or olefins, such as terminal or internal olefins), fatty alcohols, esters (e.g., wax esters, fatty acid esters (e.g., methyl or ethyl esters)), and ketones.

In some embodiments of the present invention, the higher titer of fatty acid derivatives in a particular composition is a higher titer of a particular type of fatty acid derivative (e.g., fatty alcohols, fatty acid esters, or hydrocarbons) produced by a recombinant host cell culture relative to the titer of the same fatty acid derivatives produced by a control culture of a corresponding wild-type host cell. In such cases, the fatty acid derivative compositions may comprise, for example, a mixture of the fatty alcohols with a variety of chain lengths and saturation or branching characteristics.

In other embodiments of the present invention, the higher titer of fatty acid derivatives in a particular compositions is a higher titer of a combination of different fatty acid derivatives (for example, fatty aldehydes and alcohols, or fatty acids and esters) relative to the titer of the same fatty acid derivative produced by a control culture of a corresponding wild-type host cell.

Engineering Host Cells

In some embodiments, a polynucleotide (or gene) sequence is provided to the host cell by way of a recombinant vector, which comprises a promoter operably linked to the polynucleotide sequence. In certain embodiments, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.

In some embodiments, the recombinant vector comprises at least one sequence selected from the group consisting of (a) an expression control sequence operatively coupled to the polynucleotide sequence; (b) a selection marker operatively coupled to the polynucleotide sequence; (c) a marker sequence operatively coupled to the polynucleotide sequence; (d) a purification moiety operatively coupled to the polynucleotide sequence; (e) a secretion sequence operatively coupled to the polynucleotide sequence; and (f) a targeting sequence operatively coupled to the polynucleotide sequence.

The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein.

Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy-terminus of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech, Inc., Piscataway, N.J.; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.), and pRITS (Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.

Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gni). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident 2 prophage harboring a T7 gni gene under the transcriptional control of the lacUV 5 promoter.

Suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” second edition, Cold Spring Harbor Laboratory, (1989). Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene, 69: 301-315 (1988)) and PET 11 d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., pp. 60-89 (1990)). In certain embodiments, a polynucleotide sequence of the invention is operably linked to a promoter derived from bacteriophage T5.

In one embodiment, the host cell is a yeast cell. In this embodiment, the expression vector is a yeast expression vector.

Vectors can be introduced into prokaryotic or eukaryotic cells via a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra).

For stable transformation of bacterial cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (e.g., resistance to an antibiotic) can be introduced into the host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.

Host Cells

As used herein, an engineered or recombinant “host cell” is a cell used to produce a fatty acid derivative composition as further described herein.

A host cell is referred to as an “engineered host cell” or a “recombinant host cell” if the expression of one or more polynucleotides or polypeptides in the host cell are altered or modified as compared to their expression in a corresponding wild-type (or “native”) host cell under the same conditions.

In any of the aspects of the invention described herein, the host cell can be selected from the group consisting of a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, engineered organisms thereof, or a synthetic organism. In some embodiments, the host cell is light dependent or fixes carbon. In some embodiments, the host cell has autotrophic activity.

Various host cells can be used to produce fatty acid derivatives, as described herein.

Mutants or Variants

In some embodiments, the polypeptide is a mutant or a variant of any of the polypeptides described herein. The terms “mutant” and “variant” as used herein refer to a polypeptide having an amino acid sequence that differs from a wild-type polypeptide by at least one amino acid. For example, the mutant can comprise one or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the mutant polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions.

Preferred fragments or mutants of a polypeptide retain some or all of the biological function (e.g., enzymatic activity) of the corresponding wild-type polypeptide. In some embodiments, the fragment or mutant retains at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% or more of the biological function of the corresponding wild-type polypeptide. In other embodiments, the fragment or mutant retains about 100% of the biological function of the corresponding wild-type polypeptide. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without affecting biological activity may be found using computer programs well known in the art, for example, LASERGENE™ software (DNASTAR, Inc., Madison, Wis.).

In yet other embodiments, a fragment or mutant exhibits increased biological function as compared to a corresponding wild-type polypeptide. For example, a fragment or mutant may display at least a 10%, at least a 25%, at least a 50%, at least a 75%, or at least a 90% improvement in enzymatic activity as compared to the corresponding wild-type polypeptide. In other embodiments, the fragment or mutant displays at least 100% (e.g., at least 200%, or at least 500%) improvement in enzymatic activity as compared to the corresponding wild-type polypeptide.

It is understood that the polypeptides described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide function. Whether or not a particular substitution will be tolerated (i.e., will not adversely affect desired biological function, such as carboxylic acid reductase activity) can be determined as described in Bowie et al. (Science, 247: 1306-1310 (1990)). A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Variants can be naturally occurring or created in vitro. In particular, such variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, or standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures.

Methods of making variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.

For example, variants can be prepared by using random and site-directed mutagenesis. Random and site-directed mutagenesis are described in, for example, Arnold, Curr. Opin. Biotech., 4: 450-455 (1993).

Random mutagenesis can be achieved using error prone PCR (see, e.g., Leung et al., Technique, 1: 11-15 (1989); and Caldwell et al., PCR Methods Applic., 2: 28-33 (1992)). In error prone PCR, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Briefly, in such procedures, nucleic acids to be mutagenized (e.g., a polynucleotide sequence encoding a carboxylic reductase enzyme) are mixed with PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), 0.01% gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of 94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciated that these parameters can be varied as appropriate. The mutagenized nucleic acids are then cloned into an appropriate vector, and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated.

Site-directed mutagenesis can be achieved using oligonucleotide-directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olson et al., Science, 241: 53-57 (1988). Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized (e.g., a polynucleotide sequence encoding a CAR polypeptide). Clones containing the mutagenized DNA are recovered, and the activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, for example, U.S. Pat. No. 5,965,408.

Still another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequences in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction. Sexual PCR mutagenesis is described in, for example, Stemmer, Proc. Natl. Acad. Sci., U.S.A., 91: 10747-10751 (1994).

Variants can also be created by in vivo mutagenesis. In some embodiments, random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways. Such “mutator” strains have a higher random mutation rate than that of a wild-type strain. Propagating a DNA sequence (e.g., a polynucleotide sequence encoding a CAR polypeptide) in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for in vivo mutagenesis are described in, for example, International Patent Application Publication No. WO 1991/016427.

Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double-stranded DNA molecule is replaced with a synthetic oligonucleotide “cassette” that differs from the native sequence. The oligonucleotide often contains a completely and/or partially randomized native sequence.

Recursive ensemble mutagenesis can also be used to generate variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described in, for example, Arkin et al., Proc. Natl. Acad. Sci., U.S.A., 89: 7811-7815 (1992).

In some embodiments, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described in, for example, Delegrave et al., Biotech. Res, 11: 1548-1552 (1993).

In some embodiments, variants are created using shuffling procedures wherein portions of a plurality of nucleic acids that encode distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides as described in, for example, U.S. Pat. Nos. 5,965,408 and 5,939,250.

Insertional mutagenesis is mutagenesis of DNA by the insertion of one or more bases. Insertional mutations can occur naturally, mediated by virus or transposon, or can be artificially created for research purposes in the lab, e.g., by transposon mutagenesis. When exogenous DNA is integrated into that of the host, the severity of any ensuing mutation depends entirely on the location within the host's genome wherein the DNA is inserted. For example, significant effects may be evident if a transposon inserts in the middle of an essential gene, in a promoter region, or into a repressor or an enhancer region. Transposon mutagenesis and high-throughput screening was done to find beneficial mutations that increase the titer or yield of a fatty acid derivative or derivatives.

Culture Recombinant Host Cells and Cell Cultures/Fermentation

As used herein, the term “fermentation” broadly refers to the conversion of organic materials into target substances by host cells, for example, the conversion of a carbon source by recombinant host cells into fatty acids or derivatives thereof by propagating a culture of the recombinant host cells in a media comprising the carbon source.

As used herein, the term “conditions permissive for the production” means any conditions that allow a host cell to produce a desired product, such as a fatty acid or a fatty acid derivative. Similarly, the term “conditions in which the polynucleotide sequence of a vector is expressed” means any conditions that allow a host cell to synthesize a polypeptide. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters, including but not limited to temperature ranges, levels of aeration, feed rates and media composition. Each of these conditions, individually and in combination, allows the host cell to grow. Fermentation can be aerobic, anaerobic, or variations thereof (such as micro-aerobic). Exemplary culture media include broths or gels. Generally, the medium includes a carbon source that can be metabolized by a host cell directly. In addition, enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.

For small scale production, the engineered host cells can be grown in batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide sequence encoding a CAR polypeptide. For large scale production, the engineered host cells can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, and 1,000,000 L or larger; fermented; and induced to express a desired polynucleotide sequence. Alternatively, large scale fed-batch fermentation may be carried out.

The fatty acid derivative compositions described herein are found in the extracellular environment of the recombinant host cell culture and can be readily isolated from the culture medium. A fatty acid derivative may be secreted by the recombinant host cell, transported into the extracellular environment or passively transferred into the extracellular environment of the recombinant host cell culture. The fatty acid derivative is isolated from a recombinant host cell culture using routine methods known in the art.

Products Derived from Recombinant Host Cells

As used herein, “fraction of modem carbon” or fM has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1.

Bioproducts (e.g., the fatty acid derivatives produced in accordance with the present disclosure) comprising biologically produced organic compounds, and in particular, the fatty acid derivatives produced using the fatty acid biosynthetic pathway herein, have not been produced from renewable sources and, as such, are new compositions of matter. These new bioproducts can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or ¹⁴C dating. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat. No. 7,169,588, which is herein incorporated by reference).

The ability to distinguish bioproducts from petroleum based organic compounds is beneficial in tracking these materials in commerce. For example, organic compounds or chemicals comprising both biologically based and petroleum based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum based materials. Hence, the bioproducts herein can be followed or tracked in commerce on the basis of their unique carbon isotope profile.

Bioproducts can be distinguished from petroleum based organic compounds by comparing the stable carbon isotope ratio (¹³C/¹²C) in each sample. The ¹³C/¹²C ratio in a given bioproduct is a consequence of the ¹³C/¹²C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed.

It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C3 plants (the broadleaf), C4 plants (the grasses), and marine carbonates all show significant differences in ¹³C/¹²C and the corresponding δ¹³C values. Furthermore, lipid matter of C3 and C4 plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway. Within the precision of measurement, ¹³C shows large variations due to isotopic fractionation effects, the most significant of which for bioproducts is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (i.e., the initial fixation of atmospheric CO₂). Two large classes of vegetation are those that incorporate the “C3” (or Calvin-Benson) photosynthetic cycle and those that incorporate the “C4” (or Hatch-Slack) photosynthetic cycle.

In C3 plants, the primary CO₂ fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase, and the first stable product is a 3-carbon compound. C3 plants, such as hardwoods and conifers, are dominant in the temperate climate zones.

In C4 plants, an additional carboxylation reaction involving another enzyme, phosphoenolpyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid that is subsequently decarboxylated. The CO₂ thus released is refixed by the C3 cycle. Examples of C4 plants are tropical grasses, corn, and sugar cane.

Both C4 and C3 plants exhibit a range of ¹³C/¹²C isotopic ratios, but typical values are about −7 to about −13 per mil for C4 plants and about −19 to about −27 per mil for C3 plants (see, e.g., Stuiver et al., Radiocarbon 19:355 (1977)). Coal and petroleum fall generally in this latter range. The 13C measurement scale was originally defined by a zero set by Pee Dee Belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The “613C” values are expressed in parts per thousand (per mil), abbreviated, % o, and are calculated as follows:

δ¹3C(%)=[(¹³C/¹²C)sample−(¹³C/¹²C)standard]/(¹³C/¹²C) standard×1000

Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is δ¹³C. Measurements are made on CO₂ by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45, and 46.

The compositions described herein include bioproducts produced by any of the methods described herein, including, for example, fatty aldehyde and alcohol products. Specifically, the bioproduct can have a δ¹³C of about −28 or greater, about −27 or greater, −20 or greater, −18 or greater, −15 or greater, −13 or greater, −10 or greater, or −8 or greater. For example, the bioproduct can have a δ¹³C of about −30 to about −15, about −27 to about −19, about −25 to about −21, about −15 to about −5, about −13 to about −7, or about −13 to about −10. In other instances, the bioproduct can have a δ¹³C of about −10, −11, −12, or −12.3.

Bioproducts produced in accordance with the disclosure herein, can also be distinguished from petroleum based organic compounds by comparing the amount of ¹⁴C in each compound. Because ¹⁴C has a nuclear half-life of 5730 years, petroleum based fuels containing “older” carbon can be distinguished from bioproducts which contain “newer” carbon (see, e.g., Currie, “Source Apportionment of Atmospheric Particles”, Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74, (1992)).

The basic assumption in radiocarbon dating is that the constancy of ¹⁴C concentration in the atmosphere leads to the constancy of ¹⁴C in living organisms. However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, ¹⁴C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO₂, and hence in the living biosphere, approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (¹⁴C/¹²C) of about 1.2×10-12, with an approximate relaxation “half-life” of 7-10 years. (This latter half-life must not be taken literally; rather, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric ¹⁴C since the onset of the nuclear age.)

It is this latter biospheric ¹⁴C time characteristic that holds out the promise of annual dating of recent biospheric carbon. ¹⁴C can be measured by accelerator mass spectrometry (AMS), with results given in units of “fraction of modern carbon” (fM). fM is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C. As used herein, “fraction of modern carbon” or “fM” has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1.

The compositions described herein include bioproducts that can have an fM ¹⁴C of at least about 1. For example, the bioproduct of the invention can have an fM ¹⁴C of at least about 1.01, an fM ¹⁴C of about 1 to about 1.5, an fM ¹⁴C of about 1.04 to about 1.18, or an fM ¹⁴C of about 1.111 to about 1.124.

Another measurement of ¹⁴C is known as the percent of modern carbon (pMC). For an archaeologist or geologist using ¹⁴C dates, AD 1950 equals “zero years old”. This also represents 100 pMC. “Bomb carbon” in the atmosphere reached almost twice the normal level in 1963 at the peak of thermo-nuclear weapons. Its distribution within the atmosphere has been approximated since its appearance, showing values that are greater than 100 pMC for plants and animals living since AD 1950. It has gradually decreased over time with today's value being near 107.5 pMC. This means that a fresh biomass material, such as corn, would give a ¹⁴C signature near 107.5 pMC. Petroleum based compounds will have a pMC value of zero. Combining fossil carbon with present day carbon will result in a dilution of the present day pMC content. By presuming 107.5 pMC represents the ¹⁴C content of present day biomass materials and 0 pMC represents the ¹⁴C content of petroleum based products, the measured pMC value for that material will reflect the proportions of the two component types. For example, a material derived 100% from present day soybeans would give a radiocarbon signature near 107.5 pMC. If that material was diluted 50% with petroleum based products, it would give a radiocarbon signature of approximately 54 pMC.

A biologically based carbon content is derived by assigning “100%” equal to 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all the components within the analyzed material originated either from present day biological material or petroleum based material.

A bioproduct comprising one or more fatty acid derivatives as described herein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other instances, a fatty acid derivative described herein can have a pMC of between about 50 and about 100; about 60 and about 100; about 70 and about 100; about 80 and about 100; about 85 and about 100; about 87 and about 98; or about 90 and about 95. In yet other instances, a fatty acid derivative described herein can have a pMC of about 90, 91, 92, 93, 94, or 94.2.

Screening Fatty Acid Derivative Compositions Produced by Recombinant Host Cells

To determine if conditions are sufficient to allow expression, a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48 hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow expression. For example, the host cells in the sample or the medium in which the host cells were grown can be tested for the presence of a desired product. When testing for the presence of a product, assays, such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, can be used. Recombinant host cell cultures are screened at the 96 well plate level, 1 liter and 5 liter tank level and in a 1000 L pilot plant using a GC/FID assay for “total fatty species”.

Utility of Fatty Acid Derivative Compositions

A fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides. When they are not attached to other molecules, they are known as “free” fatty acids. Fatty acids are usually produced industrially by the hydrolysis of triglycerides, with the removal of glycerol.

Palm, soybean, rapeseed, coconut oil and sunflower oil are currently the most common sources of fatty acids. The majority of fatty acids derived from such sources are used in human food products. Coconut oil and palm kernel oil (consist mainly of 12 and 14 carbon fatty acids). These are particularly suitable for further processing to surfactants for washing and cleansing agents as well as cosmetics. Palm, soybean, rapeseed, and sunflower oil, as well as animal fats such as tallow, contain mainly long-chain fatty acids (e.g., C18, saturated and unsaturated) which are used as raw materials for polymer applications and lubricants. Ecological and toxicological studies suggest that fatty acid-derived products based on renewable resources have more favorable properties than petrochemical-based substances.

Fatty aldehydes are used to produce many specialty chemicals. For example, aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which may be used as solvents, preservatives, or disinfectants. In addition, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction.

Fatty alcohols have many commercial uses. Worldwide annual sales of fatty alcohols and their derivatives are in excess of U.S. $1 billion. The shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, such as, for example, detergents. In addition, fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats.

The invention also provides a surfactant composition or a detergent composition comprising a fatty alcohol produced by any of the methods described herein. One of ordinary skill in the art will appreciate that, depending upon the intended purpose of the surfactant or detergent composition, different fatty alcohols can be produced and used. For example, when the fatty alcohols described herein are used as a feedstock for surfactant or detergent production, one of ordinary skill in the art will appreciate that the characteristics of the fatty alcohol feedstock will affect the characteristics of the surfactant or detergent composition produced. Hence, the characteristics of the surfactant or detergent composition can be selected for by producing particular fatty alcohols for use as a feedstock.

A fatty alcohol-based surfactant and/or detergent composition described herein can be mixed with other surfactants and/or detergents well known in the art. In some embodiments, the mixture can include at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or a range bounded by any two of the foregoing values, by weight of the fatty alcohol. In other examples, a surfactant or detergent composition can be made that includes at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range bounded by any two of the foregoing values, by weight of a fatty alcohol that includes a carbon chain that is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbons in length. Such surfactant or detergent compositions also can include at least one additive, such as a microemulsion or a surfactant or detergent from non-microbial sources such as plant oils or petroleum, which can be present in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range bounded by any two of the foregoing values, by weight of the fatty alcohol.

Esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl esters, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor, which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.

Hydrocarbons have many commercial uses. For example, shorter chain alkanes are used as fuels. Longer chain alkanes (e.g., from five to sixteen carbons) are used as transportation fuels (e.g., gasoline, diesel, or aviation fuel). Alkanes having more than sixteen carbon atoms are important components of fuel oils and lubricating oils. Even longer alkanes, which are solid at room temperature, can be used, for example, as a paraffin wax. In addition, longer chain alkanes can be cracked to produce commercially valuable shorter chain hydrocarbons.

Like short chain alkanes, short chain alkenes are used in transportation fuels. Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants. In addition, alkenes are used as a feedstock to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinic anhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives, and drag flow reducers.

Ketones are used commercially as solvents. For example, acetone is frequently used as a solvent, but it is also a raw material for making polymers. Ketones are also used in lacquers, paints, explosives, perfumes, and textile processing. In addition, ketones are used to produce alcohols, alkenes, alkanes, imines, and enamines.

Lubricants are typically composed of olefins, particularly polyolefins and alpha-olefins. Lubricants can either be refined from crude petroleum or manufactured using raw materials refined from crude petroleum. Obtaining these specialty chemicals from crude petroleum requires a significant financial investment as well as a great deal of energy. It is also an inefficient process because frequently the long chain hydrocarbons in crude petroleum are cracked to produce smaller monomers. These monomers are then used as the raw material to manufacture the more complex specialty chemicals.

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

EXAMPLES Example 1 Production Host Modifications—Attenuation of Acyl-CoA Dehydrogenase

This example describes the construction of a genetically engineered host cell wherein the expression of a fatty acid degradation enzyme is attenuated.

The fadE gene of E. coli MG1655 (an E. coli K strain) was deleted using the Lambda Red (also known as the Red-Driven Integration) system described by Datsenko et al., Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000), with the following modifications:

The following two primers were used to create the deletion of fadE:

Del-fadE-F (SEQ ID NO: 9) 5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATA TTGATTCCGGGGATCCGTCGACC; and Del-fadE-R (SEQ ID NO: 10) 5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTT CCTGTAGGCTGGAGCTGCTTC

The Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 (described by Datsenko et al., supra) by PCR. The PCR product was then used to transform electrocompetent E. coli MG1655 cells containing pKD46 (described in Datsenko et al., supra) that had been previously induced with arabinose for 3-4 hours. Following a 3-hour outgrowth in a super optimal broth with catabolite repression (SOC) medium at 37° C., the cells were plated on Luria agar plates containing 50 μg/mL of Kanamycin. Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the fadE gene was confirmed by PCR amplification using primers fadE-L2 and fadE-R1, which were designed to flank the E. coli fadE gene.

The fadE deletion confirmation primers were:

(SEQ ID NO: 11) fadE-L2 5′-CGGGCAGGTGCTATGACCAGGAC; and (SEQ ID NO: 12) fadE-R1 5′-CGCGGCGTTGACCGGCAGCCTGG

After the fadE deletion was confirmed, a single colony was used to remove the KmR marker using the pCP20 plasmid as described by Datsenko et al., supra. The resulting MG1655 E. coli strain with the fadE gene deleted and the KmR marker removed was named E. coli MG1655 AfadE, or E. coli MG 1655 D1.

Fatty acid derivative (“total fatty species”) production by the MG1655 E. coli strain with the fadE gene deleted was compared to fatty acid derivative production by E. coli MG1655. The data presented in FIG. 7 shows that deletion of the fadE gene did not affect fatty acid derivative production.

A number of exemplary host cell strains are described herein, examples of which are described below in Table 3.

TABLE 3 Genetic Characterization of E. coli strains. Strain Genetic Characterization DV2 MG1655 F-, λ-, ilvG-, rfb-50, rph-1, ΔfhuA::FRT, ΔfadE::FRT DV2.1 DV2 fabB::fabB[A329V] D178 DV2.1 entD::loxP_P_(T5) _(—) entD EG149 D178 insH-11::(P_(LACUV5)-V_(cho) _(—) fabV-S_(typ) _(—) (fabHDG)- S_(typ) _(—) fabA-C_(ace) _(—) fabF_FRT), iFAB138 V642 EG149 rph+ SL313 V642 lacIZ::P_(A1) _(—) ′tesA/pDG109 V668 V642 ilvG⁺ LC397 V668 lacIZ::P_(TRC) _(—) 12H08_kan SL571 V668 lacIZ:: P_(TRC) _(—) 12H08_FRT V324 D178 lacIZ::P_(TRC) _(—) tesA ALC310 D178/pALC310 V928 LC397/pV869 LC341 LC397/pLC308 V940 LC397/pV171.1 LC434 LC397/pLC274 EG442 V642 Tn7::P_(TRC)-ABR lacIZ::P_(T5O)-ABR D851 SL571 yijP::Tn5-cat/pV171.1 D859 SL571 yijP::Tn5-cat/pEP55 BD64 DV2 ifab138 iT5fadR Shu.002 Isogenic to BD64 except that it contains the T5 promoter controlling expression of the FAB138 operon Shu.034 Isogenic to shu2 except that it also contains the yijP::Tn5-cat transposon cassette ABR denotes the operon alrAadp1-fabB[A329G]-fadR, P_(T5O) is an inducible T5 promoter containing a lacO binding site, and P_(TRC-AT) is the TRC promoter with the anti-termination region removed.

Example 2 Increased Flux Through the Fatty Acid Synthesis Pathway—Acetyl CoA Carboxylase Mediated A. Fatty Ester Production.

The main precursors for fatty acid biosynthesis are malonyl-CoA and acetyl-CoA (FIG. 1). It has been suggested that these precursors limit the rate of fatty acid biosynthesis in E. coli. In this study, synthetic acc operons [Corynebacterium glutamicum accABCD (±birA)] were overexpressed and the genetic modifications led to increased acetyl-coA and malonyl-CoA production in E. coli.

In one approach, in order to increase malonyl-CoA levels, an acetyl-CoA carboxylase enzyme complex from Corynebacterium glutamicum (“C. glutamicum”) was overexpressed. Acetyl-CoA carboxylase (“acc”) consists of four discrete subunits, accA, accB, accC and accD (FIG. 3). The advantage of C. glutamicum acc is that two subunits are expressed as fusion proteins, accCB and accDA, respectively, which facilitates its balanced expression. Additionally, C. glutamicum birA, which biotinylates the accB subunit (FIG. 3). Exemplary C. glutamicum bir DNA sequences are presented as SEQ ID NO:55 and SEQ ID NO:56. A C. glutamicum bir protein sequence is presented as SEQ ID NO:57.

The synthetic operons of the C. glutamicum acc genes were cloned in the following way in OP80: Ptrc1-accDACB, Ptrc3-accDACB, Ptrc1-accCBDA and Ptrc3-CBDA. Ptrc1 and Ptrc3 are derivatives of the commonly used Ptrc promoter, which allow attenuated transcription of target genes. Note that the native sequences were amplified from the chromosomal DNA as they showed favorable codon usage (only the codon for Arg6 in accCB was changed). The C. glutamicum birA gene was codon optimized and obtained by gene synthesis. It was cloned then downstream of the acc genes in all four operon constructs. Below we refer to the operon configuration accDACB as accD- and the operon configuration accDACB+birA as accD+.

The resulting plasmids were transformed into E. coli DAM1_i377, which contains integrated copies (i) of leaderless thioesterease 'tesA and acyl-CoA synthetase fadD from E. coli and Ester synthase 9 (ES9) from Marinobacter hydrocarbonoclasticus. All genes are controlled by Ptrc promoters. The strains were grown in 5NBT media in shake flasks and were analyzed for malonyl-CoA using the method described above. FIG. 9 shows that six of the eight C. glutamicum acc±birA constructs showed elevated levels of malonyl-CoA in logarithmic phase demonstrating their functionality in E. coli. It was noted that coexpression of birA further increased malonyl-CoA levels in the ptrc1/3_accDACB strains, in particular with the plasmid containing the Ptrc3-accDACB-birA operon configuration (plasmid pAS119.50D; SEQ ID NO:62).

In order to test the effect of combining panK and acc-birA overexpression, the optimized panK gene was cloned downstream of birA in ptrc1/3_accDACB-birA. Pantothenate kinase panK (or CoaA) catalyzes the first step in the biosynthesis of coenzyme A, an essential cofactor that is involved in many reactions, e.g. the formation of acetyl-CoA, the substrate for acetyl-CoA carboxylase. The resulting plasmids were transformed into DAM1_i377, grown in 5NBT (+TVS1) media in shake flasks, and the strains were analyzed for short-chain-CoAs using the method described above.

As shown in FIG. 9, in log phase panK coexpression further increased malonyl-CoA levels and also increased acetyl-CoA levels demonstrating that panK can further increase the malonyl-CoA levels

The impact of coexpressing an acetyl-CoA carboxylase enzyme complex on fatty ester production was evaluated by expressing ester synthase 9 (SEQ ID NO:6) with and without acc genes in another E. coli production host. More specifically, plasmids OP80 (vector control), pDS57 (with ES9), pDS57-accD- (with ES9 and accDACB) or pDS57-accD+(with ES9 and accDACB-birA; SEQ ID NO:63) were transformed into E. coli strain DV2 and the corresponding transformants were selected on LB plates supplemented with 100 mg/L of spectinomycin.

Two transformants of each plasmid were independently inoculated into LB medium supplemented with 100 mg/L of spectinomycin and grown for 5-8 hours at 32C. The cultures were diluted 30-fold into a minimal medium with the following composition: 0.5 g/L NaCl, 1 mM MgSO4×7H2O, 0.1 mM CaCl2, 2 g/L NH4C1, 3 g/L KH2PO4, 6 g/L Na2HPO4, 1 mg/L thiamine, 1× trace metal solution, 10 mg/L ferric citrate, 100 mM Bis-Tris (pH7.0), 30 g/L glucose and 100 mg/L spectinomycin. After over-night growth at 32 C, the cultures were diluted 10-fold in quadruplicate into minimal medium of the same composition except that the media contained 1 g/L instead of 2 g/L NH4Cl and was supplemented with 1 mM IPTG and 2% (v/v) methanol. The resulting cultures were then grown at 32° C. in a shaker.

The production of fatty acid methyl esters (FAMEs) was analyzed by gas chromatography with flame ionization detector (GC-FID). The samples were extracted with butyl acetate in a ratio of 1:1 vol/vol. After strong vortexing, the samples were centrifuged, and the organic phase was analyzed by gas chromatography (GC). The analysis conditions were as follows: instrument: Trace GC Ultra, Thermo Electron Corporation with Flame ionization detector (FID) detector; column: DB-1 (1% diphenyl siloxane; 99% dimethyl siloxane) CO1 UFM 1/0.1/5 01 DET from Thermo Electron Corporation, phase pH 5, FT: 0.4 μm, length 5 m, id: 0.1 mm; inlet conditions: 250° C. splitless, 3.8 m 1/25 split method used depending upon sample concentration with split flow of 75 mL/m; carrier gas, flow rate: Helium, 3.0 mL/m; block temperature: 330° C.; oven temperature: 0.5 m hold at 50° C., 100° C./m to 330° C., 0.5 m hold at 330° C.; detector temperature: 300° C.; injection volume: 2 μL; run time/flow rate: 6.3 m/3.0 mL/m (splitless method), 3.8 m/1.5 mL/m (split 1/25 method), 3.04 m/1.2 mL/m (split 1/50 method).

FAMEs produced are shown in FIG. 10. The expression of ES9 by itself in E. coli DV2 led to FAME production above the control DV20P80. Coexpression of the C. glutamicum acetyl-CoA carboxylase complex led to an approx. 1.5-fold increase in FAMEs and the additional expression of the C. glutamicum biotin protein ligase led to an approx. 5-fold increase in FAMEs. These results suggest that the increased supply of malonyl-CoA improves the ability of ES9 to convert intermediates of the fatty acid biosynthetic machinery to fatty acid methyl esters in E. coli.

B. Fatty Alcohol Production

The impact of coexpressing an acetyl-CoA carboxylase enzyme complex on Fatty alcohol production was evaluated by expressing the Acyl-ACP reductase (AAR) from Synechococcus elongatus with and without acc genes in E. coli DV2. The accD+operon configuration was selected as it gave the best results when coexpressed with ester synthase (see previous example).

The accDABC-birA operon was cloned downstream from the aar gene in pLS9185, a pCL1920 derivative) using Infusion technology, the resulting plasmid was transformed into E. coli DV2 and the corresponding transformants were selected on LB plates supplemented with 100 mg/L of spectinomycin.

Fatty alcohols produced are shown in FIG. 11. The coexpression of AAR and accD+led to a ca. 1.5-fold increase in fatty alcohol titers as compared to the AAR only control (PLS9185). The data were reproducible (triplicate samples were shown). These results demonstrate that increasing malonyl-CoA levels lead to improved fatty acid production when this acyl-ACP reductase is used.

In addition, Example 3 describes co-expression of acc genes together with entire fab operons.

Example 3 Increased Flux Through the Fatty Acid Synthesis Pathway—iFABs A. Fatty Acid Derivative Production

Strategies to increase the flux through the fatty acid synthesis pathway in recombinant host cells include both overexpression of native E. coli fatty acid biosynthesis genes and expression of exogenous fatty acid biosynthesis genes from different organisms in E. coli.

In this study, exogenous fatty acid biosynthesis genes from different organisms were combined in the genome of E. coli DV2. E. coli DV2 has the following genetic characterization: F-, λ-, ilvG-, rfb-50, rph-1, ΔfhuA::FRT, ΔfadE::FRT.

Sixteen strains containing iFABs 130-145 were evaluated. The detailed structure of iFABs 130-145 is presented in iFABs Table 4, below.

TABLE 4 Components found in iFABs 130-145. Abbreviation Full Description St_fabD Salmonella typhimurium fabD gene nSt_fabH Salmonella typhimurium fabH gene with the native RBS sSt_fabH Salmonella typhimurium fabH gene with a synthetic RBS Cac_fabF Clostridium acetobutylicum (ATCC824) fabF gene St_fabG Salmonella typhimurium fabG gene St_fabA Salmonella typhimurium fabA gene St_fabZ Salmonella typhimurium fabZ gene BS_fabI Bacillus subtilis fabI gene BS_fabL Bacillus subtilis fabL gene Vc_FabV Vibrio chorlerae fabV gene Ec_FabI Escherichia coli fabI gene

Each “iFAB” comprises various components in the following order: BS_fabI, BS_FabL, Vc_FabV, or Ec_FabI. All constructs contain St_H, St_D, and St_G, yet half of them have a synthetic RBS in front of St_H. All constructs contain either St_fabA or St_fabZ.

All constructs include Cac_fabF. See Table 4, below for the specific composition of iFABs 130-145.

TABLE 4 Compostion of iFABs 130-145. Strain Name BS_fabI BS_fabL Vc_fabV Ec_fabI nSt_fabH sSt_fabH St_fabD St_fabG St_fabA St_fabZ Cac_fabF DV2ifab130 1 0 0 0 1 0 1 1 1 0 1 DV2iFab131 1 0 0 0 1 0 1 1 0 1 1 DV2iFab132 1 0 0 0 0 1 1 1 1 0 1 DV2iFab133 1 0 0 0 0 1 1 1 0 1 1 DV2ifab134 0 1 0 0 1 0 1 1 1 0 1 DV2iFab135 0 1 0 0 1 0 1 1 0 1 1 DV2iFab136 0 1 0 0 0 1 1 1 1 0 1 DV2iFab137 0 1 0 0 0 1 1 1 0 1 1 DV2iFab138 0 0 1 0 1 0 1 1 1 0 1 DV2iFab139 0 0 1 0 1 0 1 1 0 1 1 DV2iFab140 0 0 1 0 0 1 1 1 1 0 1 DV2iFab141 0 0 1 0 0 1 1 1 0 1 1 DV2ifab142 0 0 0 1 1 0 1 1 1 0 1 DV2iFab143 0 0 0 1 1 0 1 1 0 1 1 DV2iFab144 0 0 0 1 0 1 1 1 1 0 1 DV2iFab145 0 0 0 1 0 1 1 1 0 1 1

The plasmid pCL-WT TRC WT TesA was transformed into each of the strains shown above and a fermentation was run in FA2 media with 20 hours from induction to harvest at both 32° C. and 37° C. Data for production of “Total Fatty Species” from duplicate plate screens is shown in FIGS. 12A and 12B.

From this screen the best construct was determined to be DV2 with iFAB138. The iFAB138 construct was transferred into strain D178 to make strain EG149. This strain was used for further engineering. The sequence of iFAB138 in the genome of EG149 is presented as SEQ ID NO:19. (LOCUS integrated_pDS138 8029 bp ds-DNA linear15-JUL-2010).

B. Fatty Ester Production

A full synthetic fab operon was integrated into the E. coli chromosome and evaluated for increased FAME production by expression in E. coli DAM1 pDS57. In addition, four synthetic acc operons from Corynebaterium glutamicum were coexpressed and evaluated for improved FAME productivity. Several strains were obtained that produced FAMEs at a faster rate and higher titers.

Sixteen different fab operons were constructed (either assembled in vitro or as plasmid-based intermediates) as summarized in Table 5. The fab operons were put under the control of the lacUV5 promoter and integrated into the IS5-11 site of E. coli DAM1. These strains were named ifab130 to 145. They were transformed either with pDS57 (containing ester synthase 377) or pDS57 coexpressing different versions of acc operons, see above) for evaluation of FAME production. Exemplary plasmids are described in Table 5.

TABLE 5 Genotype of integrated fab operons. Strain Genotype of additional fab operon DAM1- IS5-11::PlacUV5 BsfabI (natRBS) StfabHDG StfabA ifab130 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabI (natRBS) StfabHDG StfabZ ifab131 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabI (synRBS) StfabHDG ifab132 StfabART CacfabF::F DAM1- IS5-11::PlacUV5 BsfabI (synRBS) StfabHDG StfabZ ifab133 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabL (natRBS) StfabHDG StfabA ifab134 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabL (natRBS) StfabHDG StfabZ ifab135 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabL (synRBS) StfabHDG StfabA ifab136 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabL (synRBS) StfabHDG StfabZ ifab137 CacfabF::FRT DAM1- IS5-11::PlacUV5 VcfabV (natRBS) StfabHDG StfabA ifab138 CacfabF::FRT DAM1- IS5-11::PlacUV5 VcfabV (natRBS) StfabHDG StfabZ ifab139 CacfabF::FRT DAM1- IS5-11::PlacUV5 VcfabV (synRBS) StfabHDG StfabA ifab140 CacfabF::FRT DAM1- IS5-11::PlacUV5 VcfabV (synRBS) StfabHDG StfabZ ifab141 CacfabF::FRT DAM1- IS5-11::PlacUV5 EcfabI (natRBS) StfabHDG StfabA ifab142 CacfabF::FRT DAM1- IS5-11::PlacUV5 EcfabI (natRBS) StfabHDG StfabZ ifab143 CacfabF::FRT DAM1- IS5-11::PlacUV5 EcfabI (synRBS) StfabHDG StfabA ifab144 CacfabF::FRT DAM1- IS5-11::PlacUV5 EcfabI (synRBS) StfabHDG StfabZ ifab145 CacfabF::FRT Bs: Bacillus subtilis; St: Salmonella typhimurium; Cac: Clostridium acetobutylicum; Vc: Vibrio cholera; Ec: Escherichia coli .

TABLE 6 Plasmids containing ester synthase ES9 (from Marinobacter hydrocarbonclasticus) and synthetic acc operons (from Corynebactrium glutamicum) Plasmid Genes pTB.071 pDS57-accCBDA pTB.072 pDS57-ES9-accCBDA-birA pTB.073 pDS57-ES9-accDACB pTB.074 pDS57-ES9-accDACB-birA pDS57 = pCL_ptrc-ES9

The DAM1 ifab strains were analyzed in 96-well plates (4NBT medium), shake flasks (5NBT medium) and in fermenters at 32° C. The best results were obtained in 96-well plates and in shake flasks, where several DAM1 ifab strains with pDS57-acc-birA plasmids showed higher FAME titers. In particular, DAM1 ifab131, ifab135, ifab137, ifab138 and ifab143 with pDS57-accDACB-birA showed 20-40% improved titers indicating that in these strains a higher flux through the fatty acid pathway was achieved, which apparently resulted in a better product formation rate (these results were reproducible in several independent experiments).

It was also observed that the FAMEs produced by some of the outperforming DAM1 ifab/acc strains showed a shift towards higher chain length. In particular, DAM1 ifab138 pDS57-accDACB-birA showed a significantly higher C16 and C18 to C8-C14 FAME ratio than the control. These results suggest that a stronger pull by tesA/fadD/WS377 may further improve FAME production.

C. Effect of Overexpressing fabH and fabI on Fatty Acid Methyl Ester (FAME) Production

Strategies to increase the flux through the fatty acid synthesis pathway in recombinant host cells include both overexpression of native fatty acid biosynthesis genes and expression of heterologous fatty acid biosynthesis genes. FabH and fabI are two fatty acid biosynthetic enzymes that have been shown to be feedback inhibited. A study was conducted to determine if FabH and FabI might be limiting the rate of FAME production.

FabH and fabI homologues (from E. coli, B. subtilis, Acinetobacter baylyi ADP1, Marinobacter aquaeoli VT8, and Rhodococcus opacus) were overexpressed as a synthetic operon and evaluated in E. coli DAM1 pDS57 (a strain observed to be a good FAME producer).

In one approach, fabHfabI operons were constructed from organisms that accumulate waxes (A. baylyi, M. aquaeoli) or triacylglycerides (R. opacus) and integrated into the chromosome of E. coli DAM1 pDS57. In a related approach, a synthetic acc operons from C. glutamicum were co-expressed (as described in Example 2, above).

Eleven different fabHI operons were constructed (assembled in vitro) as summarized in Table 7. The fabHI operons were put under the control of IPTG inducible lacUV5 promoter and integrated into the IS5-11 site of E. coli DAM1. These strains were named as shown in the table below. They were transformed either with pDS57 (containing ester synthase 377) or pDS57 coexpressing different versions of acc operons for evaluation of FAME production.

TABLE 7 Genotype of integrated fabHI operons Strain Genotype of additional fab operon plasmid StEP117 DAM 1 IS5-11 ::PlacUV5 (synRBS) EcfabH (synRBS) bsfabI::kan pDS57 StEP118 DAM 1 IS5-11 ::PlacUV5 (synRBS) EcfabH (synRBS) BsfabL::kan pDS57 StEP127 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ecfabH (ecRBS) bsfabI::kan pDS57 StEP128 DAM 1 IS5-11 ::PlacUV5 (ecRBS) EcfabH (ecRBS) BsfabL::kan pDS57 StEP129 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ADP1fabH (ecRBS) ADP1fabI::kan pDS57 StEP130 DAM 1 IS5-11 ::PlacUV5 (synRBS) ADP1fabH (synRBS) ADP1fabI::kan pDS57 StEP131 DAM 1 IS5-11 ::PlacUV5 (synRBS) VT8fabH1 (synRBS) VT8fabI::kan pDS57 StEP132 DAM 1 IS5-11 ::PlacUV5 (synRBS) VT8fabH2 (synRBS) VT8fabI::kan pDS57 StEP133 DAM 1 IS5-11 ::PlacUV5 (ecRBS) VT8fabH1 (synRBS) VT8fabI::kan pDS57 StEP134 DAM 1 IS5-11 ::PlacUV5 (ecRBS) VT8fabH2 (synRBS) VT8fabI::kan pDS57 StEP151 DAM 1 IS5 11::PlacUV5 (synRBS)RofabI (synRBS) RofabH::kan pDS57 StEP153 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ADP1fabH (ecRBS) ADP1fabI::kan pDS57-accCBDA StEP154 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ADP1fabH (ecRBS) ADP1fabI::kan pDS57-accDACB StEP155 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ADP1fabH (ecRBS) ADP1fabI::kan pDS57-accCBDA-birA StEP156 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ADP1fabH (ecRBS) ADP1fabI::kan pDS57-accDACB-birA StEP157 DAM 1 IS5-11 ::PlacUV5 (synRBS) ecfabH (synRBS) bsfabI::kan pDS57-accCBDA StEP158 DAM 1 IS5-11 ::PlacUV5 (synRBS) ecfabH (synRBS) bsfabI::kan pDS57-accCBDA-birA StEP159 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ecfabH (synRBS) bsfabI::kan pDS57-accCBDA StEP160 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ecfabH (synRBS) bsfabI::kan pDS57-accCBDA-birA StEP161 DAM 1 IS5-11 ::PlacUV5 (ecRBS) VT8fabH1 (synRBS) VT8fabI::kan pDS57-accCBDA StEP162 DAM 1 IS5-11 ::PlacUV5 (ecRBS) VT8fabH1 (synRBS) VT8fabI::kan pDS57-accCBDA-birA StEP163 DAM 1 IS5-11 ::PlacUV5 (ecRBS) VT8fabH2 (synRBS) VT8fabI::kan pDS57-accCBDA StEP164 DAM 1 IS5-11 ::PlacUV5 (ecRBS) VT8fabH2 (synRBS) VT8fabI::kan pDS57-accCBDA-birA Bs: Bacillus subtilis; Ec: Escherichia coli, ADP1: Acinetobacter sp. ADP1, VT8: Marinobacter aquaeolei VT8, Ro; Rhodococcus opacus B4

The DAM1 ifabHI strains were analyzed in 96-well plates (4NBT medium), shake flasks (5NBT medium) and in fermenters at 32° C.

In shake flask, a number of the ifabHI strains carrying pDS57 plasmid performed better than the control DAM1 pDS57strain, reaching 10 to 15% higher FAME titers (FIG. 13). Additional increase in FAME titers was obtained when ifabHI strains were transformed with pDS57-acc-birA plasmids, in particular an increase of 50% in FAME titers was observed in strain StEP156 (DAM1 5-11::UV5(ecRBS)ADP1fabH (ecRBS)ADP1fabI pDS57-accDACB-birA) (FIG. 14).

Some of the ifabHI strains were also run in fermenters, where an increase in FAME titers, specific productivity and yield were also observed (FIG. 15), indicating that in these strains a higher flux through the fatty acid pathway was achieved, which resulted in a better product formation rate. In particular stEP129 (DAM1 5-11::UV5(ecRBS)ADP1fabH (ecRBS)ADP1fabI pDS57) showed higher FAME titers and yield in several independent fermentation runs. Other combinations of fabH and fabI may be used to achieve similar effects. Although FAME is exemplified here, this approach increases the flux through the fatty acid biosynthetic pathway and is therefore a useful approach to increase production of any fatty acid derivative.

D. Effect of Inserting a Strong Promoter in Front of Operon FAB138 on Fatty Acid Methyl Ester (FAME) Production

The lacUV5 promoter of FAB138 was replaced by a T5 promoter leading to higher levels of expression of FAB138, as confirmed by mRNA analysis. The expression of FAB138 from the T5 promoter resulted in a higher titer, yield and productivity of fatty esters.

Strain BD64 is DV2 ifab138 iT5_fadR. Strain shu.002 is isogenic to strain BD64 except that it contains the T5 promoter controlling expression of the FAB138 operon.

TABLE 8 Primers used to Generate iT5 138 Cassette and Verify its Insertion in New Strains SEQ Primer ID Name NO Sequence DG405 20 TTGTCCATCTTTATATAATTTGGGGGTAGGGTGTT CTTTATGTAAAAAAAACgtttTAGGATGCATATGG CGGCC DG406 21 GATAAATCCACGAATTTTAGGTTTGATGATCATTG GTCTCCTCCTGCAGGTGCGTGTTCGTCGTCATCGC AATTG DG422 22 ACTCACCGCATTGGTGTAGTAAGGCGCACC DG423 23 TGAATGTCATCACGCAGTTCCCAGTCATCC DG744 24 CCATCTTCTTTGTACAGACGTTGACTGAACATG DG749 24 GCACCATAGCCGTAATCCCACAGGTTATAG oTREE047 26 TGTCATTAATGGTTAATAATGTTGA

Primers DG405 and DG406 were used to amplify a cat-loxP and T5 promoter cassette adding 50 bp homology to each end of the PCR product, such that it could be integrated into any strain replacing the lacUV5 promoter regulating expression of the FAB138 operon. The cat-loxP-T5 promoter was transformed into BD64/pKD46 strain. Transformants were recovered on LB+chloramphenicol plates at 37° C. overnight, patched to a fresh LB+chloramphenicol plate, and verified by colony PCR using primers DG422 and DG423. Plasmid pJW168 was transformed into strain BD64 i-cat-loxP-T5_(—)138 and selected on LB+carbenicillin plates at 32° C. In order to remove the cat marker, expression of the cre-recombinase was induced by IPTG. The plasmid pHW168 was removed by growing cultures at 42° C. Colonies were patched on LB+chloramphenicol and LB+carbenicillin to verify loss of pJW168 and removal of cat marker, respectively. The colony was also patched into LB as a positive control, all patched plates were incubated at 32° C. The removal of the cat marker was confirmed by colony PCR using primers DG422 and DG423. The resulting PCR product was verified by sequencing with primers EG744, EG749 and oTREE047, the strain was called shu.002. FIGS. 16A and B provides a map of the strains generated.

FIG. 16 shows the FAB138 locus: a diagram of the cat-loxP-T5 promoter integrated in front of FAB138 (FIG. 16A) and a diagram of the iT5_(—)138 promoter region (FIG. 16B).

The sequence of the cat-loxP-T5 promoter integrated in front of FAB138 with 50 base pair of homology shown in each side of cat-loxP-T5 promoter region is presented as SEQ ID NO:1 and the sequence of the iT5_(—)138 promoter region with 50 base pair homology in each side is presented as SEQ ID NO:2.

There are a number of conditions that can lead to increased fatty acid flux. In this example increased fatty acid flux was achieved by altering the promoter strength of operon FAB138. The expression of FAB138 from the T5 promoter was beneficial, however, when this promoter change was combined with the insertion of yijP::Tn5 cassette further improvements were observed in titer, yield and productivity of fatty acid esters and other fatty acid derivatives. (See Example 5).

Example 4 Increasing the Amount of Free Fatty Acid (FFA) Product by Repairing the Rph and ilvG Mutations

The ilvG and rph mutations were corrected in this strain resulting in higher production of FFA. Strains EG149 (D178 is 5-11::iFAB138) and V668 (EG149 rph+ilvG+) were transformed with pCL-tesA obtained from D191. Fermentation was run at 32° C. in FA2 media for 40 hours to compare the FFA production of strains D178, EG149, and V668 with pCL-tesA. Fermentation and extraction was run according to a standard FALC fermentation protocol exemplified by the following.

A frozen cell bank vial of the selected E. coli strain was used to inoculate 20 mL of LB broth in a 125 mL baffled shake flask containing spectinomycin antibiotic at a concentration of 115 μg/mL. This shake flask was incubated in an orbital shaker at 32° C. for approximately six hours, then 1.25 mL of the broth was transferred into 125 mL of low P FA2 seed media (2 g/L NH₄C1, 0.5 g/L NaCl, 3 g/L KH₂PO₄, 0.25 g/L MgSO₄-7H2O, 0.015 g/L mM CaCl₂-2H2O, 30 g/L glucose, 1 mL/L of a trace minerals solution (2 g/L of ZnCl₂. 4H₂O, 2 g/L of CaCl₂. 6H₂O, 2 g/L of Na₂MoO₄. 2H₂O, 1.9 g/L of CuSO₄.5H₂O, 0.5 g/L of H₃BO₃, and 10 mL/L of concentrated HCl), 10 mg/L of ferric citrate, 100 mM of Bis-Tris buffer (pH 7.0), and 115 μg/mL of spectinomycin), in a 500 mL baffled Erlenmeyer shake flask, and incubated on a shaker overnight at 32° C.

100 mL of this low P FA2 seed culture was used to inoculate a 5 L Biostat Aplus bioreactor (Sartorius BBI), initially containing 1.9 L of sterilized F1 bioreactor fermentation medium. This medium is initially composed of 3.5 g/L of KH₂PO₄, 0.5 g/L of (NH₄)₂SO₄, 0.5 g/L of MgSO₄ heptahydrate, 10 g/L of sterile filtered glucose, 80 mg/L ferric citrate, 5 g/L Casamino acids, 10 mL/L of the sterile filtered trace minerals solution, 1.25 mL/L of a sterile filtered vitamin solution (0.42 g/L of riboflavin, 5.4 g/L of pantothenic acid, 6 g/L of niacin, 1.4 g/L of pyridoxine, 0.06 g/L of biotin, and 0.04 g/L of folic acid), and the spectinomycin at the same concentration as utilized in the seed media. The pH of the culture was maintained at 6.9 using 28% w/v ammonia water, the temperature at 33° C., the aeration rate at 1 lpm (0.5 v/v/m), and the dissolved oxygen tension at 30% of saturation, utilizing the agitation loop cascaded to the DO controller and oxygen supplementation. Foaming was controlled by the automated addition of a silicone emulsion based antifoam (Dow Corning 1410).

A nutrient feed composed of 3.9 g/L MgSO₄ heptahydrate and 600 g/L glucose was started when the glucose in the initial medium was almost depleted (approximately 4-6 hours following inoculation) under an exponential feed rate of 0.3 hr-1 to a constant maximal glucose feed rate of 10-12 g/L/hr, based on the nominal fermentation volume of 2 L. Production of fatty alcohol in the bioreactor was induced when the culture attained an OD of 5 AU (approximately 3-4 hours following inoculation) by the addition of a 1M IPTG stock solution to a final concentration of 1 mM. The bioreactor was sampled twice per day thereafter, and harvested approximately 72 hours following inoculation.

A 0.5 mL sample of the well-mixed fermentation broth was transferred into a 15 mL conical tube (VWR), and thoroughly mixed with 5 mL of butyl acetate. The tube was inverted several times to mix, then vortexed vigorously for approximately two minutes. The tube was then centrifuged for five minutes to separate the organic and aqueous layers, and a portion of the organic layer transferred into a glass vial for gas chromatographic analysis.

Correcting the rph and ilvG mutations resulted in a 116% increase in the FFA production of the base strain with pCL-tesA. As seen in FIG. 17, V668/pCL-tesA produces more FFA than the D178/pCL-tesA, or the EG149/pCL-tesA control. Since FFA is a precursor to the LS9 products, higher FFA production is a good indicator that the new strain can product higher levels of LS9 products.

It has been demonstrated that expression of many genes, not limited to, fabA, B, Z, G, H, D, and fadR can lead to increased fatty acid production. Further strain improvements are likely to result in higher titers, yields and productivity of fatty acid derivatives such as FALC by recombinant host cells.

Example 5 Increased Production of Fatty Acid Derivatives by Transposon Mutagenesis—yijP A. Fatty Alcohol Production

To improve the titer, yield, productivity of fatty alcohol production by E. coli, transposon mutagenesis and high-throughput screening was carried out and beneficial mutations were sequenced. A transposon insertion in the yijP strain was shown to improve the strain's fatty alcohol yield in both shake flask and fed-batch fermentations.

The SL313 strain produces fatty alcohols. The genotype of this strain is provided in Table **.

The genotype of this strain is MG1655 (ΔfadE::FRT ΔfhuA::FRT fabBA329V ΔentD::T5-entD ΔinsH-11:: PlacUV5 fab138 rph+lacI::PA1_tesA) containing the plasmid pDG109 (pCL1920_PTRC_carBopt_(—)12H08_alrAadp1_fabB[A329G]_fadR).

Briefly, transposon mutagenesis was carried out by preparation of transposon DNA was prepared by cloning a DNA fragment into the plasmid EZ-Tn5™ pMOD™<R6K ori/MCS> (Epicentre Biotechnologies). The DNA fragment contains a T5 promoter and the cat gene flanked by loxP sites. The resulting plasmid was named p100.38 and the sequence is listed in Appendix I. This plasmid was digested with PshAI restriction enzyme, incubated with EZ-Tn5™ Transposase enzyme (Epicentre Biotechnologies), and electroporated into electrocompetent SL313 cells as per the manufacturer's instructions. The resulting colonies contained the transposon DNA inserted randomly into the chromosome of SL313.

Transposon clones were then subjected to high-throughput screening to measure production of fatty alcohols. Briefly, colonies were picked into deep-well plates containing LB, grown overnight, inoculated into fresh LB and grown for 3 hours, inoculated into fresh FA-2.1 media, grown for 16 hours, then extracted using butyl acetate. The crude extract was derivatized with BSTFA (N,O-bis[Trimethylsilyl]trifluoroacetamide) and analyzed using GC/FID. Spectinomycin (100 ug/mL) was included in all media to maintain selection of the pDG109 plasmid.

Hits were selected by choosing clones that produced a similar total fatty species as the control strain SL313, but that had a higher percent of fatty alcohol species and a lower percent of free fatty acids than the control. Strain 68F11 was identified as a hit and was validated in a shake flask fermentation, according to the shake flask fermentation method described below. A comparison of transposon hit 68F11 to control strain SL313 indicated that 68F11 produces a higher percentage of fatty alcohol species than the control, while both strains produce similar titers of total fatty species.

A single colony of hit 68F11, named LC535, was sequenced to identify the location of the transposon insertion. Sequencing was performed according to previous transposon IDFs. Briefly, genomic DNA was purified from a 10 mL overnight LB culture using the kit ZR Fungal/Bacterial DNA MiniPrep™ (Zymo Research) according to the manufacturer's instructions. The purified genomic DNA was sequenced outward from the transposon using primers internal to the transposon:

DG150 (SEQ ID NO: 27) 5′-GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGCCTG-3′ DG131 (SEQ ID NO: 28) 5′-GAGCCAATATGCGAGAACACCCGAGAA-3′

Strain LC535 was determined to have a transposon insertion in the yijP gene (FIG. 18). yijP encodes a conserved inner membrane protein whose function is unclear. The yijP gene is in an operon and co-transcribed with the ppc gene, encoding phosphoenolpyruvate carboxylase, and the yijO gene, encoding a predicted DNA-binding transcriptional regulator of unknown function. Promoters internal to the transposon likely have effects on the level and timing of transcription of yijP, ppc and yijO, and may also have effects on adjacent genes frwD, pflC, pfld, and argE. Promoters internal to the transposon cassette are shown, and may have effects on adjacent gene expression.

Strain LC535 was evaluated in a fed-batch fermentation on two different dates. Both fermentations demonstrated that LC535 produced fatty alcohols with a higher yield than control SL313, and the improvement was 1.3-1.9% absolute yield based on carbon input.

The yijP transposon cassette was further evaluated in a different strain V940, which produces fatty alcohol at a higher yield than strain SL313. The yijP::Tn5-cat cassette was amplified from strain LC535 using primers:

LC277 (SEQ ID NO: 29) 5′-CGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGCA G-3′ LC278 (SEQ ID NO: 30) 5′-GGAATTGCCACGGTGCGGCAGGCTCCATACGCGAGGCCAGGTTATCC AACG-3′ This linear DNA was electroporated into strain SL571 and integrated into the chromosome using the lambda red recombination system. Colonies were screened using primers outside the transposon region:

(SEQ ID NO: 31) DG407 5′-AATCACCAGCACTAAAGTGCGCGGTTCGTTACCCG-3′ (SEQ ID NO: 32) DG408 5′-ATCTGCCGTGGATTGCAGAGTCTATTCAGCTACG-3′

A colony with the correct yijP transposon cassette was transformed with the production plasmid pV171.1 to produce strain D851. D851 (V940 yijP::Tn5-cat) was tested in a shake-flask fermentation against isogenic strain V940 that does not contain the yijP transposon cassette. The result of this fermentation showed that the yijP transposon cassette confers production of a higher percent of fatty alcohol by the D851 strain relative to the V940 strain and produces similar titers of total fatty species as the V940 control strain.

Strain D851 was evaluated in a fed-batch fermentation on two different dates. Data from these fermentations is shown in Table 9 which illustrates that in 5-liter fed-batch fermentations, strains with the yijP::Tn5-cat transposon insertion had an increased total fatty species (“FAS”) yield and an increase in percent fatty alcohol (“FALC”).

The terms “total fatty species” and “total fatty acid product” may be used interchangeably herein with reference to the amount of fatty alcohols, fatty aldehydes and free fatty acids, as evaluated by GC-FID as described in International Patent Application Publication WO 2008/119082. The same terms may be used to mean fatty esters and free fatty acids when referring to a fatty ester analysis. As used herein, the term “fatty esters” includes beta hydroxy esters.

TABLE 9 Effect of yijp transposon insertion on titer and yield of FAS and FALC. FAS FAS Percent FALC Strain Titer Yield FALC Yield V940 68 g/L 18.7% 95.0% 17.8% D851 70 g/L 19.4% 96.1% 18.6% V940 64 g/L 18.4% 91.9% 16.9% D851 67 g/L 19.0% 94.0% 17.8%

Shake Flask Fermentation Method

To assess production of fatty acid esters in tank a glycerol vial of desired strain was used to inoculate 20 mL LB+spectinomycin in shake flask and incubated at 32° C. for approximately six hours. 4 mL of LB culture was used to inoculate 125 mL Low PFA Seed Media (below), which was then incubated at 32° C. shaker overnight. 50 mL of the overnight culture was used to inoculate 1 L of Tank Media. Tanks were run at pH 7.2 and 30.5° C. under pH stat conditions with a maximum feed rate of 16 g/L/hr (glucose or methanol).

TABLE 10 Low P FA Seed Media Component Concentration NH4Cl 2 g/L NaCl 0.5 g/L KH2PO4 1 g/L MgSO4—7H2O 0.25 g/L CaCl2—2H2O 0.015 g/L Glucose 20 g/L TM2 Trace Minerals solution 1 mL/L Ferric citrate 10 mg/L Bis Tris buffer (pH 7.0) 100 mM Spectinomycin 115 mg/L

TABLE 11 Tank Media Component Concentration (NH4)2SO4 0.5 g/L KH2PO4 3.0 g/L Ferric Citrate 0.034 g/L TM2 Trace Minerals 10 mL/L Solution Casamino acids 5 g/L Post sterile additions MgSO4—7H2O 2.2 g/L Trace Vitamins 1.25 mL/L Solution Glucose 5 g/L Inoculum 50 mL/L

Further studies suggest that the improved titer and yield of FAS and FALC in strains with the yijP transposon insertion is due to reduction in the activity of phosphoenolpyruvate carboxylase (ppc). A ppc enzyme assay was carried out in-vitro in the following strains to evaluate this hypothesis.

-   -   1) Δppc=DG14 (LC942 Δppc::cat-sacB/pLC56)     -   2) wt-ppc=DG16 (LC942/pLC56)     -   3) yijP::Tn5=DG18 (LC942 yijP::Tn5-cat/pCL56)

Ppc activity was measured in cells grown in a shake flask fermentation (as detailed above) and harvested 12-16 hours after induction. Approximately 5 mL of cells were centrifuged and the cell paste was suspended in BugBuster Protein Extraction Reagent (Novagen) with a protease inhibitor cocktail solution. The cell suspension was incubated with gentle shaking on a shaker for 20 min. Insoluble cell debris was removed by centrifugation at 16,000×g for 20 min at 4° C. followed by transferring the supernatant to a new tube. Ppc activity in the cell lysate was determined by a coupling reaction with citrate synthase using following reaction mixture: 0.4 mM acetyl-CoA, 10 mM phosphoenolpyruvate, 0.5 mM monobromobimane, 5 mM MgCl₂, 10 mM NaHCO₃, and 10 units citrate synthase from porcine heart in 100 mM Tris-HCl (pH 8.0). The formation of CoA in the reaction with citrate synthase using oxaloacetate and acetyl-CoA was monitored photometrically using fluorescent derivatization of CoA with monobromobimane.

The Ppc assay results showed that the yijP::Tn5-cat transposon cassette decreased the Ppc activity in the cell. The results also indicate that the highest yield of fatty alcohol production requires a level of Ppc expression lower than the wild-type level.

Proteomics data was also collected to assess the abundance of the Ppc protein in two strains with and without the yijP::Tn5-cat transposon cassette. Protein samples were collected from strains V940 and D851 grown in bioreactors under standard fatty alcohol production conditions. Samples were taken at three different time points: 32, 48, 56 hours and prepared for analysis.

Sample collection and protein isolation was carried out as follows:

-   -   1. 20 ml of fermentation broth were collected from each         bioreactor at each time point. Samples were quenched with         ice-cold PBS and harvested by centrifugation (4500 rpm/10 min)         at 4° C. Cell pellet was washed with ice-cold PBS and         centrifuged one more time and stored at −80° C. for further         processing.     -   2. Total protein extraction was performed using a French press         protocol. Briefly, cell pellets were resuspended in 7 ml of         ice-cold PBS and French pressed at 2000 psi twice to ensure         complete lysing of the bacteria. Samples were centrifuged for 20         min at 10000 rpm at 4° C. to separate non-lysed cells and cell         debris from the protein fraction. Total protein concentration of         clear lysate was determined using BCA Protein Assay Reagent.         Samples were diluted to 2 mg proteins/ml concentration and         frozen at −80° C.     -   3. Samples were resuspended in the appropriate buffer and         trypsinized overnight at 37° C. and lyophilized. Fragmented         protein samples were labeled with isotopically enriched         methylpiperazine acetic acid at room temperature for 30 min.         Labeled samples were separated using cation exchange liquid         chromatography and subjected to mass spectroscopy analysis using         an ion trap mass spectrometer. Raw data was normalized using         background subtraction and bias correction.

Proteomics data showed a significant reduction in the relative abundance of Ppc protein in D851 strain when compared to V940 at 36 hours and 48 hours). These data show that the yijP::Tn5-cat transposon cassette results in a significant reduction in Ppc abundance in the cell. This suggests that the observed benefits to fatty alcohol production by strains harboring the yijP::Tn5-cat transposon hit is due to reducing the amount of Ppc protein.

These results suggest that altering ppc activity can improve the yield of fatty acid derivatives. There are a number of ways to alter the expression of the ppc gene, and the yijP transposon insertion is one way to accomplish this. While the mechanism is not part of the invention, if the effect of reducing phosphoenolpyruvate carboxylase activity is to limit the flow of carbon through the TCA cycle, one could achieve similar results by decreasing the activity of citrate synthase (gltA) or slowing the TCA cycle by decreasing the activity of any of the enzymes involved in the TCA cycle.

B. Fatty Ester Production

Additional strains with a transposon insertion in yijP were evaluated for production of fatty acid esters. Strains containing a transposon insertion in yijP were shown to produce higher yields of fatty acid esters and maintain the glucose utilization rate for a longer time in tanks.

A strain designated, “shu.010” was developed which is isogenic to strain BD64 except that it contains the yijP::Tn5-cat transposon cassette. The cassette containing the yijP::(Tn5) transposon DNA was amplified from strain DG851 using primers DG408 and DG407 (Table 12). The cassette was transformed into BD64/pKD46. Transformants were recovered on LB+chloramphenicol plates at 37° C. overnight, patched to a fresh LB+chloramphenicol plate, and verified by colony PCR using primers DG131, DG407, and DG408.

TABLE 12 Primers used to amplify yijP::Tn5 cassette and verify its insertion in new strains SEQ ID Name NO: Sequence Description DG131 28 GAGCCAATATGCGAGAACACCCGAGAA Primer in Tn5 DG407 31 AATCACCAGCACTAAAGTGCGCGGTTCGTTACCCG Primer 568 bp of Tn5 insertion site DG408 32 ATCTGCCGTGGATTGCAGAGTCTATTCAGCTACG Forward primer 464 bp of Tn5 insertion site Expected in transformants Wild-type Primer Pair Product Size: 572 bp DG131/DG408 Product size: 1101 bp DG407/DG408

Plasmid pKEV022 was transformed into shu.010. After selection in LB+spectinomycin plates, one colony was selected and called shu.015. Strain shu.015 was grown in tanks using standard conditions (see Appendix I for media and tank conditions). The tank performance of shu.015 was compared to strains KEV006.1 (BD64 pKEV018) and KEV075 (BD64 pKEV022) for Total Fatty Acid Product, Total Product Yield and glucose utilization rate.

The yield of total fatty acid products for all strains was similar, however, shu.015 was able to sustain higher glucose utilization rates for a longer time than either KEV006.1 or KEV075, suggesting that yijP::Tn5 was responsible for the improvement.

Example 6 Increased Flux Through the Fatty Acid Synthesis Pathway—Acyl Carrier Protein (ACP) Mediated Fatty Alcohol Production

When terminal pathway enzymes from sources other than E. coli are expressed in E. coli as the heterologous host to convert fatty acyl-ACPs to products, limitations may exist in the recognition, affinity and/or turnover of the recombinant pathway enzyme towards the E. coli fatty acyl-ACPs. Note that although ACP proteins are conserved to some extent in all organisms, their primary sequence can differ significantly.

To test this hypothesis the acp genes from several cyanobacteria were cloned downstream from the Synechococcus elongatus PCC7942 acyl-ACP reductase (AAR) present in pLS9-185, which is a pCL1920 derivative (3-5 copies/cell). In addition, the sfp gene (Accession no. X63158; SEQ ID NO:53) from Bacillus subtilis, encoding a phosphopantetheinyl transferase with broad substrate specificity, was cloned downstream of the respective acp genes. This enzyme is involved in conversion of the inactive apo-ACP to the active holo-ACP. The plasmids constructed are described in Table 13.

TABLE 13 Plasmids coexpressing cyanobacterial ACP with and without B. subtilis sfp downstream from S. elongatus PCC7942 AAR. ACP SEQ ID Base NO. (DNA/ Without plasmid ACP Source Polypeptide) sfp With sfp pLS9-185 Synechococcus 49/50 pDS168 pDS168S elongatus 7942 pLS9-185 Synechocystis 45/46 pDS169 not available sp. 6803 pLS9-185 Prochlorococcus 47/48 pDS170 pDS170S marinus MED4 pLS9-185 Nostoc punctiforme 43/44 pDS171 pDS171S 73102 pLS9-185 Nostoc sp. 7120 51/52 pDS172 pDS172S

All the acp genes were cloned with a synthetic RBS into the EcoRI site immediately downstream of the aar gene in pLS9-185 using InFusion technology. The EcoRI site was reconstructed downstream of the acp gene. Similarly, the B. subtilis sfp gene was InFusion cloned into this EcoRI site along with a synthetic RBS. All plasmids were transformed into E. coli MG1655 DV2. The control for these experiments was the expression of AAR alone (pLS9-185).

The results from standard shake flask fermentation experiments are shown in FIG. 19. Significant improvement in fatty alcohol titers were observed in strains containing the plasmids pDS171S, pDS 172S, pDS 168 and pDS169demonstrating that ACP overexpression can be beneficial for fatty alcohol production, in this case presumably by aiding in the recognition, affinity and/or turnover of acyl-ACPs by the heterologous terminal pathway enzyme. (See Table 13 for the source of the ACPs and presence or absence of sfp.)

Fatty Acid Production.

In order to evaluate if the overexpression of an ACP can also increase free fatty acid production, one cyanobacterial ACP gene with sfp was amplified from pDS171s (Table 13) and cloned downstream from 'tesA into a pCL vector. The resulting operon was under the control of the Ptrc3 promoter, which provides slightly lower transcription levels than the Ptrc wildtype promoter. The construct was cloned into E. coli DV2 and evaluated for fatty acid production. The control strain contained the identical plasmid but without cyanobacterial ACP and B. subtilis sfp.

The results from a standard microtiter plate fermentation experiment are shown in FIG. 20. Significant improvement in fatty acid titer was observed in the strain coexpressing the heterologous ACP demonstrating that ACP overexpression can be beneficial for fatty acid production, in this case presumably by increasing the flux through the fatty acid biosynthetic pathway.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Preferred embodiments of this invention are described herein. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

TABLE 14 Table of Sequences. SEQ ID Designation Sequence 1 cat-loxP-T5 (in TTGTCCATCTTTATATAATTTGGGGGTAGGGTGTTCTTTATGTAAAAAAAACgtttTAGGATGCATATG front of iFAB138) GCGGCCGCataacttcgtataGCATACATtatacgaagttaTCTAGAGTTGCATGCCTGCAGGtccgct tattatcacttattcaggcgtagcAaccaggcgtttaagggcaccaataactgccttaaaaaaattacg ccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcac aaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgccca tggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcaccc agggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcg atgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagct caccgtctttcattgccatacggaattccggatgagcattcatcaggcgggcaagaatgtgaataaagg ccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtct ggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatat caacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataact caaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaa cgtctcattttcgccaaaagttggcccagggcttcccggtatcaacagggacaccaggatttatttatt ctgcgaagtgatcttccgtcacaggtatttattcGACTCTAGataacttcgtataGCATACATTATACG AAGTTATGGATCCAGCTTATCGATACCGTCaaacAAATCATAAAAAATTTATTTGCTTTcaggaaaatt tttctgTATAATAGATTCAATTGCGATGACGACGAACACGCACCTGCAGGAGGAGACCAATGATCATCA AACCTAAAATTCGTGGATTTATC 2 T5 (in front of TTGTCCATCTTTATATAATTTGGGGGTAGGGTGTTCTTTATGTAAAAAAAACgtttTAGGATGCATATG iFAB138) GCGGCCGCataacttcgtataGCATACATTATACGAAGTTATGGATCCAGCTTATCGATACCGTCaaac AAATCATAAAAAATTTATTTGCTTTcaggaaaatttttctgTATAATAGATTCAATTGCGATGACGACG AACACGCACCTGCAGGAGGAGACCAATGATCATCAAACCTAAAATTCGTGGATTTATC 3 AlrA Acinetobacter MSNHQIRAYAAMQAGEQVVPYQFDAGELKAHQVEVKVEYCGLCHSDLSVINNEWQSSVYPAVAGHEIIG sp. M-1 TIIALGSEAKGLKLGQRVGIGWTAETCQACDPCIGGNQVLCTGEKKATIIGHAGGFADKVRAGWQWVIP LPDDLDPESAGPLLCGGITVLDPLLKHKIQATHHVGVIGIGGLGHIAIKLLKAWGCEITAFSSNPDKTE ELKANGADQVVNSRDAQAIKGTRWKLIILSTANGTLNVKAYLNTLAPKGSLHFLGVTLEPIPVSVGAIM GGAKSVTSSPTGSPLALRQLLQFAARKNIAPQVELFPMSQLNEAIERLHSGQARYRIVLKADFD 4 AlrAadp1 MATTNVIHAYAAMQAGEALVPYSFDAGELQPHQVEVKVEYCGLCHSDVSVLNNEWHSSVYPVVAGHEVI GTITQLGSEAKGLKIGQRVGIGWTAESCQACDQCISGQQVLCTGENTATIIGHAGGFADKVRAGWQWVI PLPDELDPTSAGPLLCGGITVFDPILKHQIQATHHVAVIGIGGLGHMAIKLLKAWGCEITAFSSNPNKT DELKAMGADHVVNSRDDAEIKSQQGKFDLLLSTVNVPLNWNAYLNTLAPNGTFHFLGVVMEPIPVPVGA LLGGAKSLTASPTGSPAALRKLLEFAARKNIAPQIEMY 5 yjgB atgTCGATGATAAAAAGCTATGCCGCAAAAGAAGCGGGCGGCGAACTGGAAGTTTATGAGTACGATCCC GGTGAGCTGAGGCCACAAGATGTTGAAGTGCAGGTGGATTACTGCGGGATCTGCCATTCCGATCTGTCG ATGATCGATAACGAATGGGGATTTTCACAATATCCGCTGGTTGCCGGGCATGAGGTGATTGGGCGCGTG GTGGCACTCGGGAGCGCCGCGCAGGATAAAGGTTTGCAGGTCGGTCAGCGTGTCGGGATTGGCTGGACG GCGCGTAGCTGTGGTCACTGCGACGCCTGTATTAGCGGTAATCAGATCAACTGCGAGCAAGGTGCGGTG CCGACGATTATGAATCGCGGTGGCTTTGCCGAGAAGTTGCGTGCGGACTGGCAATGGGTGATTCCACTG CCAGAAAATATTGATATCGAGTCCGCCGGGCCGCTGTTGTGCGGCGGTATCACGGTCTTTAAACCACTG TTGATGCACCATATCACTGCTACCAGCCGCGTTGGGGTAATTGGTATTGGCGGGCTGGGGCATATCGCT ATAAAACTTCTGCACGCAATGGGATGCGAGGTGACAGCCTTTAGTTCTAATCCGGCGAAAGAGCAGGAA GTGCTGGCGATGGGTGCCGATAAAGTGGTGAATAGCCGCGATCCGCAGGCACTGAAAGCACTGGCGGGG CAGTTTGATCTCATTATCAACACCGTCAACGTCAGCCTCGACTGGCAGCCCTATTTTGAGGCGCTGACC TATGGCGGTAATTTCCATACGGTCGGTGCGGTTCTCACGCCGCTGTCTGTTCCGGCCTTTACGTTAATT GCGGGCGATCGCAGCGTCTCTGGTTCTGCTACCGGCACGCCTTATGAGCTGCGTAAGCTGATGCGTTTT GCCGCCCGCAGCAAGGTTGCGCCGACCACCGAACTGTTCCCGATGTCGAAAATTAACGACGCCATCCAG CATGTGCGCGACGGTAAGGCGCGTTACCGCGTGGTGTTGAAAGCCGATTTTtga 6 ES9 of Marinobacter 1 MKRLGTLDAS WLAVESEDTP MHVGTLQIFS LPEGAPETFL RDMVTRMKEA GDVAPPWGYK hydrocarbonoclasticus 61 LAWSGFLGRV IAPAWKVDKD IDLDYHVRHS ALPRPGGERE LGILVSRLHS NPLDFSRPLW DSM8789 protein 121 ECHVIEGLEN NRFALYTKMH HSMIDGISGV RLMQRVLTTD PERCNMPPPW TVRPHQRRGA 181 KTDKEASVPA AVSQAMDALK LQADMAPRLW QAGNRLVHSV RHPEDGLTAP FTGPVSVLNH 241 RVTAQRRFAT QHYQLDRLKN LAHASGGSLN DIVLYLCGTA LRRFLAEQNN LPDTPLTAGI 301 PVNIRPADDE GTGTQISFMI ASLATDEADP LNRLQQIKTS TRRAKEHLQK LPKSALTQYT 361 MLLMSPYILQ LMSGLGGRMR PVFNVTISNV PGPEGTLYYE GARLEAMYPV SLIAHGGALN 421 ITCLSYAGSL NFGFTGCRDT LPSMQKLAVY TGEALDELES LILPPKKRAR TRK 7 ES8 of Marinobacter MTPLNPTDQLFLWLEKRQQPMHVGGLQLFSFPEGAPDDYVAQLADQLRQK hydrocarbonoclasticus TEVTAPFNQRLSYRLGQPVWVEDEHLDLEHHFRFEALPTPGRIRELLSFV DSM8789(GenBank SAEHSHLMDRERPMWEVHLIEGLKDRQFALYTKVHHSLVDGVSAMRMATR Accession No. MLSENPDEHGMPPIWDLPCLSRDRGESDGHSLWRSVTHLLGLSDRQLGTI ABO21021) protein PTVAKELLKTINQARKDPAYDSIFHAPRCMLNQKITGSRRFAAQSWCLKR IRAVCEAYGTTVNDVVTAMCAAALRTYLMNQDALPEKPLVAFVPVSLRRD DSSGGNQVGVILASLHTDVQDAGERLLKIHHGMEEAKQRYRHMSPEEIVN YTALTLAPAAFHLLTGLAPKWQTFNVVISNVPGPSRPLYWNGAKLEGMYP VSIDMDRLALNMTLTSYNDQVEFGLIGCRRTLPSLQRMLDYLEQGLAELE LNAGL 8 ester synthase AtfA1 MKALSPVDQLFLWLEKRQQPMHVGGLQLFSFPEGAGPKYVSELAQQMRDY from Alcanivorax CHPVAPFNQRLTRRLGQYYWTRDKQFDIDHHFRHEALPKPGRIRELLSLV borkumensis SK2 SAEHSNLLDRERPMWEAHLIEGIRGRQFALYYKIHHSVMDGISAMRIASK (YP.sub.-694462) TLSTDPSEREMAPAWAFNTKKRSRSLPSNPVDMASSMARLTASISKQAAT protein VPGLAREVYKVTQKAKKDENYVSIFQAPDTILNNTITGSRRFAAQSFPLP RLKVIAKAYNCTINTVVLSMCGHALREYLISQHALPDEPLIAMVPMSLRQ DDSTGGNQIGMILANLGTHICDPANRLRVIHDSVEEAKSRFSQMSPEEIL NFTALTMAPTGLNLLTGLAPKWRAFNVVISNIPGPKEPLYWNGAQLQGVY PVSIALDRIALNITLTSYVDQMEFGLIACRRTLPSMQRLLDYLEQSIREL EIGAGIK 9 Del-fadE-F AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATATTGATTCCGGGGATCCGTCGACC 10 Del-fadE-R AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTTCCTGTAGGCTGGAGCTGCTTC 11 fadE-L2 CGGGCAGGTGCTATGACCAGGAC 12 fadE-R1 CGCGGCGTTGACCGGCAGCCTGG 13 D+ F1 CCTTGGCATTGGCAATTTGAGAATTCGAGGAGGAAAACTAAATGACCATTTCCTCACCTT 14 D+ R1 TTTTGTTCGGGCCCAAGCTTTTATTGCAAACGCAGATGCGTGATTTCACCCGCATTCAGC 15 D+ R2 CGGGCCCAAGCTTCGAATTCTTATTGCAAACGCAGATGCGTGATTTCACCCGCATTCAGC 16 D+ F3 GAATAGCGCCGTCGACGAGGAGGAAAACTAAATGACCATTTCCTCACCTTTGATTGACGT 17 D+ R3 TGATGATGATGATGGTCGACTTATTGCAAACGCAGATGCGTGATTTCACCCGCATTCAGC 18 D+ operon GAGGAGGAAAACTAAATGACCATTTCCTCACCTTTGATTGACGTCGCCAACCTTCCAGACATCAACACC ACTGCCGGCAAGATCGCCGACCTTAAGGCTCGCCGCGCGGAAGCCCATTTCCCCATGGGTGAAAAGGCA GTAGAGAAGGTCCACGCTGCTGGACGCCTCACTGCCCGTGAGCGCTTGGATTACTTACTCGATGAGGGC TCCTTCATCGAGACCGATCAGCTGGCTCGCCACCGCACCACCGCTTTCGGCCTGGGCGCTAAGCGTCCT GCAACCGACGGCATCGTGACCGGCTGGGGCACCATTGATGGACGCGAAGTCTGCATCTTCTCGCAGGAC GGCACCGTATTCGGTGGCGCGCTTGGTGAGGTGTACGGCGAAAAGATGATCAAGATCATGGAGCTGGCA ATCGACACCGGCCGCCCATTGATCGGTCTTTACGAAGGCGCTGGCGCTCGTATTCAGGACGGCGCTGTC TCCCTGGACTTCATTTCCCAGACCTTCTACCAAAACATTCAGGCTTCTGGCGTTATCCCACAGATCTCC GTCATCATGGGCGCATGTGCAGGTGGCAACGCTTACGGCCCAGCTCTGACCGACTTCGTGGTCATGGTG GACAAGACCTCCAAGATGTTCGTTACCGGCCCAGACGTGATCAAGACCGTCACCGGCGAGGAAATCACC CAGGAAGAGCTTGGCGGAGCAACCACCCACATGGTGACCGCTGGTAACTCCCACTACACCGCTGCGACC GATGAGGAAGCACTGGATTGGGTACAGGACCTGGTGTCCTTCCTCCCATCCAACAATCGCTCCTACGCA CCGATGGAAGACTTCGACGAGGAAGAAGGCGGCGTTGAAGAAAACATCACCGCTGACGATCTGAAGCTC GACGAGATCATCCCAGATTCCGCGACCGTTCCTTACGACGTCCGCGATGTCATCGAATGCCTCACCGAC GATGGCGAATACCTGGAAATCCAGGCAGACCGCGCAGAAAACGTTGTTATTGCATTCGGCCGCATCGAA GGCCAGTCCGTTGGCTTTGTTGCCAACCAGCCAACCCAGTTCGCTGGCTGCCTGGACATCGACTCCTCT GAGAAGGCAGCTCGCTTCGTCCGCACCTGCGACGCGTTCAACATCCCAATCGTCATGCTTGTCGACGTC CCCGGCTTCCTCCCAGGCGCAGGCCAGGAGTACGGTGGCATTCTGCGTCGTGGCGCAAAGCTGCTCTAC GCATACGGCGAAGCAACCGTTCCAAAGATCACCGTCACCATGCGTAAGGCTTACGGCGGAGCGTACTGC GTGATGGGTTCCAAGGGCTTGGGCTCTGACATCAACCTTGCATGGCCAACCGCACAGATCGCCGTCATG GGCGCTGCTGGCGCAGTTGGATTCATCTACCGCAAGGAGCTCATGGCAGCTGATGCCAAGGGCCTCGAT ACCGTAGCTCTGGCTAAGTCCTTCGAGCGCGAGTATGAAGACCACATGCTCAACCCGTACCACGCTGCA GAACGTGGCCTGATCGACGCCGTGATCCTGCCAAGCGAAACCCGCGGACAGATTTCCCGCAACCTTCGC CTGCTCAAGCACAAGAACGTCACTCGCCCTGCTCGCAAGCACGGCAACATGCCACTGTAAGGAGGAAAA CTAAATGTCAGTCGAGACTCGCAAGATCACCAAGGTTCTTGTCGCTAACCGTGGTGAGATTGCAATCCG CGTGTTCCGTGCAGCTCGAGATGAAGGCATCGGATCTGTCGCCGTCTACGCAGAGCCAGATGCAGATGC ACCATTCGTGTCATATGCAGACGAGGCTTTTGCCCTCGGTGGCCAAACATCCGCTGAGTCCTACCTTGT CATTGACAAGATCATCGATGCGGCCCGCAAGTCCGGCGCCGACGCCATCCACCCCGGCTACGGCTTCCT CGCAGAAAACGCTGACTTCGCAGAAGCAGTCATCAACGAAGGCCTGATCTGGATTGGACCTTCACCTGA GTCCATCCGCTCCCTCGGCGACAAGGTCACCGCTCGCCACATCGCAGATACCGCCAAGGCTCCAATGGC TCCTGGCACCAAGGAACCAGTAAAAGACGCAGCAGAAGTTGTGGCTTTCGCTGAAGAATTCGGTCTCCC AATCGCCATCAAGGCAGCTTTCGGTGGCGGCGGACGTGGCATGAAGGTTGCCTACAAGATGGAAGAAGT CGCTGACCTCTTCGAGTCCGCAACCCGTGAAGCAACCGCAGCGTTCGGCCGCGGCGAGTGCTTCGTGGA GCGCTACCTGGACAAGGCACGCCACGTTGAGGCTCAGGTCATCGCCGATAAGCACGGCAACGTTGTTGT CGCCGGAACCCGTGACTGCTCCCTGCAGCGCCGTTTCCAGAAGCTCGTCGAAGAAGCACCAGCACCATT CCTCACCGATGACCAGCGCGAGCGTCTCCACTCCTCCGCGAAGGCTATCTGTAAGGAAGCTGGCTACTA CGGTGCAGGCACCGTTGAGTACCTCGTTGGCTCCGACGGCCTGATCTCCTTCCTCGAGGTCAACACCCG CCTCCAGGTGGAACACCCAGTCACCGAAGAGACCACCGGCATCGACCTGGTCCGCGAAATGTTCCGCAT CGCAGAAGGCCACGAGCTCTCCATCAAGGAAGATCCAGCTCCACGCGGCCACGCATTCGAGTTCCGCAT CAACGGCGAAGACGCTGGCTCCAACTTCATGCCTGCACCAGGCAAGATCACCAGCTACCGCGAGCCACA GGGCCCAGGCGTCCGCATGGACTCCGGTGTCGTTGAAGGTTCCGAAATCTCCGGACAGTTCGACTCCAT GCTGGCAAAGCTGATCGTTTGGGGCGACACCCGCGAGCAGGCTCTCCAGCGCTCCCGCCGTGCACTTGC AGAGTACGTTGTCGAGGGCATGCCAACCGTTATCCCATTCCACCAGCACATCGTGGAAAACCCAGCATT CGTGGGCAACGACGAAGGCTTCGAGATCTACACCAAGTGGATCGAAGAGGTTTGGGATAACCCAATCGC ACCTTACGTTGACGCTTCCGAGCTCGACGAAGATGAGGACAAGACCCCAGCACAGAAGGTTGTTGTGGA GATCAACGGCCGTCGCGTTGAGGTTGCACTCCCAGGCGATCTGGCACTCGGTGGCACCGCTGGTCCTAA GAAGAAGGCCAAGAAGCGTCGCGCAGGTGGTGCAAAGGCTGGCGTATCCGGCGATGCAGTGGCAGCTCC AATGCAGGGCACTGTCATCAAGGTCAACGTCGAAGAAGGCGCTGAAGTCAACGAAGGCGACACCGTTGT TGTCCTCGAGGCTATGAAGATGGAAAACCCTGTGAAGGCTCATAAGTCCGGAACCGTAACCGGCCTTAC TGTCGCTGCAGGCGAGGGTGTCAACAAGGGCGTTGTTCTCCTCGAGATCAAGTAATCTAGAGGAGGAAA ACTAAATGAATGTTGACATTAGCCGCTCTCGTGAACCGTTGAACGTGGAACTGTTGAAAGAAAAACTGC TGCAGAACGGTGATTTCGGTCAAGTGATCTACGAGAAGGTCACCGGCTCTACCAATGCGGACCTGCTGG CTCTGGCGGGCAGCGGCGCTCCAAACTGGACCGTCAAGACTGTTGAATTTCAGGACCACGCCCGTGGCC GTCTGGGTCGTCCGTGGAGCGCACCGGAGGGTTCCCAAACCATCGTCAGCGTTCTGGTCCAACTGAGCA TTGATCAGGTGGACCGTATTGGTACGATCCCGCTGGCCGCAGGCTTGGCTGTTATGGATGCGCTGAATG ATCTGGGCGTGGAGGGTGCAGGCCTGAAATGGCCGAACGATGTTCAGATCCACGGTAAGAAGTTGTGCG GTATTCTGGTTGAAGCAACCGGCTTCGACTCCACTCCGACCGTGGTTATCGGTTGGGGTACGAATATCT CGTTGACGAAAGAAGAGCTGCCGGTCCCGCACGCGACCAGCCTGGCCCTGGAGGGTGTTGAAGTTGACC GTACGACGTTCCTGATTAACATGCTGACCCATCTGCATACCCGTCTGGATCAGTGGCAGGGTCCGTCTG TGGACTGGCTGGATGACTATCGCGCGGTTTGTAGCAGCATTGGCCAAGATGTGCGTGTCCTGCTGCCTG GTGACAAAGAGCTGCTGGGCGAGGCGATTGGCGTGGCGACCGGTGGTGAGATCCGTGTGCGCGACGCCA GCGGCACGGTCCACACGCTGAATGCGGGTGAAATCACGCATCTGCGTTTGCAATAA 19 iFAB138 TGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCGA ACTGCAGGTCGACGGATCCCCGGAATATTTAAATCATTTGTACTTTTTGAACAGCAGAGTCGCATTATG GCCACCGAAGCCCAGGCTGTTGGACAGAACGTAGTTGACTTCTGCATTACGGCCCTCGTTAGGAACGTA ATCCAGGTCGCATTCCGGATCCGCCTCTTTGTAGCCGATGGTCGGCGGAATGAAACCCTCTTCAATAGC TTTGGCACAGATAATCGCTTCGACTGCACCGCCAGCGCCCAGCAGGTGGCCGGTCATGCTCTTGGTGCT AGACACCGGCACTTTGTAGGCGTATTCACCCAGGACCGTCTTGATCGCTTGGGTTTCGAAGCTGTCATT GTACGCCGTGCTCGTACCGTGCGCGTTGATATAGGAAATGTCCTCTGGGCGGACATTATCTTCTTCCAT TGCCAGTTTCATTGCACGTGCACCACCTTCACCATTCGGCGCTGGGCTCGTGATATGATATGCGTCGCA GGTCGCACCATAGCCAACGATCTCGGCATAGATTTTGGCACCACGCTTCAGCGCGTGCTCCAACTCTTC CAAGATAACGATACCGCTGCCCTCGCCCATCACAAAACCGCTGCGATCCTTATCGAACGGGATGCTGGC GCGCTTCGGGTCCTCAGATTTGGTCACGGCCTTCATCGAGGCAAAACCCGCCAGGCTCAACGGGGTGAT ACCTGCTTCGCTACCACCAGAGATCATAACGTCGCTATAACCAAACTTAATGTTACGGAAGGACTCACC AATGCTGTTGTTCGCGCTCGCACATGCGGTGACAATGGTCGTGCAAATACCTTTAGCGCCATAACGAAT CGCCAGATTACCGCTTGCCATATTCGCAATGATCATCGGAATAGTCATAGGGCTCACACGACCCGGACC TTTGGTAATCAGCTTTTCATCCTGCTTCTCAATGGTGCCGATGCCGCCAATGCCGCTACCAACAATGAC GCCGAAACGATTCTTATCAATCGACTCCAGGTCCAGTTTGCTGTCCTTGATTGCCTCATCCGCCGCAAC GATCGCAAACTGGCTAAAACGGTCCATACGGTTCGCCTCACGCTTGTCGATAAAGTCCTCCGGGGTGAA GTCCTTCACTTCGGCAGCCAGCTTAACTTTGAAATCGGTTGCGTCAAACGCTTTGATCTTGTCAATGCC ACATTTACCCTCTTTGATGCTGCACCAGAAGCTATCAGCGTTGTTACCCACCGGCGTCACTGCACCAAT ACCCGTAATGACAACGCGGCGATTCATtttgttgcctccttTTAgaacgcggaagtatcctggaacaaa ccgactttcaaatcgtgtgcggtatagatcaggcgaccatccaccagaacctcaccgtccgccaggccc atgatcaggcgacggtttacgatacgtttgaaatgaatacgataggtgactttcctggctgtcggcaga acctggccggtaaatttcacttcgcccacgcccagagcgcggcctttgccttcgccgcccaaccagccc aggtagaatcccaccaattgccacatagcatccagacccagacaaccgggcatcaccggatcgccgata aagtggcatccgaagaaccatagatccggattgatatccagctcggcttcgacatagcctttgtcgaaa ttgccgcccgtttcggtcatcttaacgacgcggtccatcatcagcatgttcggtgcagggagttgcggc cctttagcgccaaacagttcaccacgaccagaggcaagaaggtcttcttttgtataggattcgcgttta tctaccatgttttatgtaaaccttaaaaTTAAACCATGTACATTCCGCCGTTGACGTGCAGAGTCTCAC CAGTGATGTAACTCGCTTCGTCAGAGGCTAAAAATGCAACCGCACTGGCGATTTCCTGAGCGCCGCCGA GGCGACCCGCAGGCACCTGCGCCAGGATACCCGCACGCTGATCGTCAGACAGCGCACGCGTCATGTCCG TTTCAATAAAACCCGGAGCCACAACATTGACAGTAATACCACGGGACGCAACTTCACGCGCCAGTGATT TACTGAAACCGATCAGGCCCGCTTTCGCCGCAGCGTAGTTTGCCTGACCTGCATTTCCCATGGTACCAA CCACAGAACCAATAGTGATAATGCGACCACAACGCTTTTTCATCATAGCGCGCATTACCGCTTTTGACA GGCGGAAAACGGATGATAAGTTGGTTTCGATAATATCGTTCCACTCATCATCTTTCATTCGCATCAACA GATTATCACGAGTGATACCGGCATTATTAACCAGGATATCCACTTCACCAAATTCTGCGCGAATATTTT CCAGAACAGATTCAATAGATGCAGGATCGGTCACATTCAACATCAAACCTTTCCCGTTAGCACCTAAAT AGTCGCTAATGTTCTTCGCACCATTTTCACTGGTCGCAGTCCCGATAACTTTCGCGCCGCGGGCAACGA GAGTCTCTGCAATTGCGCGGCCTATGCCACGGCTTGCACCAGTCACCAGCGCAATCTTTCCTTCAAAGC TCATGGTTTTCCTCTTTTATTGCGTAAGTGCCGCAGACAGCGCCGCCGGCTCGTTCAGCGCCGACGCTG TCAGGGTGTCGACAATACGTTTCGTCAGACCAGTGAGGACTTTACCTGGACCCACTTCATAAAGATGTT CAACGCCCTGCGCCGCGATAAATTCCACGCTCTTCGTCCACTGTACCGGATTGTACAACTGGCGAACCA GCGCATCGCGGATAGCGGCGGCATCGGTTTCACATTTCACGTCAACGTTGTTCACTACCGGCACCGTTG GCGCGCTAAAGGTAATTTTGGCTAATTCAACCGCCAGCTTATCTGCCGCTGGTTTCATCAGCGCGCAGT GCGACGGTACGCTCACCGGCAGCGGCAGCGCGCGTTTCGCGCCAGCGGCTTTACAGGCTGCGCCCGCAC GTTCTACCGCCTCTTTATGCCCGGCGATAACCACCTGTCCCGGCGAGTTAAAGTTAACCGGCGAAACAA CCTGCCCTTCGGCAGATTCTTCACAGGCTTTAGCAATAGAGGCATCATCCAGCCCGATGATCGCAGACA TGCCGCCAGTGCCTTCCGGAACCGCTTCCTGCATGAATTTACCGCGCATTTCCACCAGACGAACGGCAT CAGCAAAGTTGATGACGCCAGCGCAAACCAGCGCGGAATATTCGCCCAGGCTGTGACCTGCCATTAACG CAGGCATTTTACCGCCCTGCTGCTGCCAAACGCGCCAAAGCGCGACGGAAGCGGTTAATAACGCCGGCT GCGTCTGCCAGGTTTTATTCAGTTCTTCCGCTGGACCTTGCTGGGTGAGCGCCCACAGATCATATCCCA GAGCCGCAGAAGCTTCAGCAAACGTTTCTTCTACGATAGGGTAATTTGCCGCCATCTCGGCCAACATCC CAACGCTCTGAGAACCCTGACCGGGGAACACAAATGCAAATTGCGTCATGTTTAAATCCTTATACTAGA AACGAATCAGCGCGGAGCCCCAGGTGAATCCACCCCCGAAGGCTTCAAGCAATACCAGCTGACCGGCTT TAATTCGCCCGTCACGCACGGCTTCATCCAGCGCGCACGGCACAGAAGCCGCGGAGGTATTGCCGTGCC TGTCCAGCGTGACGACGACATTGTCCATCGACATGCCGAGTTTTTTCGCTGTCGCGCTAATGATACGCA GGTTAGCCTGATGCGGCACCAGCCAATCGAGTTCTGAGCGATCCAGGTTATTAGCCGCCAGCGTCTCAT CGACAATATGCGCCAGTTCAGTGACCGCCACTTTAAAGACTTCATTGCCCGCCATTGTCAGGTAAATCG GGTTATCCGGATTTACGCGATCGGCATTCGGCAGGGTCAGTAATTCACCGTAACGGCCATCGGCATGAA GATGAGTGGAGATAATACCCGGTTCTTCAGAAGCGCTCAGTACGGCCGCGCCTGCGCCATCGCCGAAAA TAATGATCGTACCGCGATCGCCAGGATCGCAAGTGCGGGCTAATACATCGGAACCGACCACCAGCGCGT GTTTAACCGCGCCGGATTTAACGTACTGGTCGGCGATGCTTAACGCGTAGGTGAAACCTGCGCACGCTG CCGCGACATCAAACGCCGGGCAACCTTTAATACCGAGCATACTTTGAATCTGACATGCCGCGCTTGGAA ATGCATGCGTTGCTGATGTGGTAGCCACCACAATCAAGCCAATTTGGTCTTTATCGATCCCCGCCATCT CAATCGCGCGATTCGCAGCGGTAAAGCCCATCGTCGCGACAGTTTCATTCGGCGCGGCGATATGGCGTT TACGAATACCTGTACGAGTGACAATCCACTCGTCAGAGGTCTCAACCATTTTTTCCAGATCGGCGTTAG TCCGCACTTGTTCGGGCAGATAGCTGCCAGTACCAATAATCTTCGTATACATGTACGCTCAGTCACTaa aTTACTCGATATCAATCACATCAAATTCGACTTCTGGATTGACGTCAGCATCGTAATCAATGCCTTCAA TGCCAAAGCCAAACAGCTTGATGAACTCTTCTTTGTACATGTCGTAATCGGTCAGCTCACGCAGGTTCT CTGTGGTGATTTGTGGCCACAGATCACGGCAGTGCTGCTGAATGTCATCACGCAGTTCCCAGTCATCCA AACGCAGACGATTGTGATCATCCACTTCCGGCGCTGAACCATCT 20 DG405 TTGTCCATCTTTATATAATTTGGGGGTAGGGTGTTCTTTATGTAAAAAAAACgtttTAGGATGCATATG GCGGCC 21 DG406 GATAAATCCACGAATTTTAGGTTTGATGATCATTGGTCTCCTCCTGCAGGTGCGTGTTCGTCGTCATCG CAATTG 22 DG422 ACTCACCGCATTGGTGTAGTAAGGCGCACC 23 DG423 TGAATGTCATCACGCAGTTCCCAGTCATCC 24 DG744 CCATCTTCTTTGTACAGACGTTGACTGAACATG 25 DG749 GCACCATAGCCGTAATCCCACAGGTTATAG 26 oTREE047 TGTCATTAATGGTTAATAATGTTGA 27 DG150 GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGCCTG 28 DG131 GAGCCAATATGCGAGAACACCCGAGAA 29 LC277 CGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGCAG 30 LC278 GGAATTGCCACGGTGCGGCAGGCTCCATACGCGAGGCCAGGTTATCCAACG 31 DG407 AATCACCAGCACTAAAGTGCGCGGTTCGTTACCCG 32 DG408 ATCTGCCGTGGATTGCAGAGTCTATTCAGCTACG 33 carA MTIETREDRFNRRIDHLFETDPQFAAARPDEAISAAAADPELRLPAAVKQILAGYADRPALGKRAVEFV TDEEGRTTAKLLPRFDTITYRQLAGRIQAVTNAWHNHPVNAGDRVAILGFTSVDYTTIDIALLELGAVS VPLQTSAPVAQLQPIVAETEPKVIASSVDFLADAVALVESGPAPSRLVVFDYSHEVDDQREAFEAAKGK LAGTGVVVETITDALDRGRSLADAPLYVPDEADPLTLLIYTSGSTGTPKGAMYPESKTATMWQAGSKAR WDETLGVMPSITLNFMPMSHVMGRGILCSTLASGGTAYFAARSDLSTFLEDLALVRPTQLNFVPRIWDM LFQEYQSRLDNRRAEGSEDRAEAAVLEEVRTQLLGGRFVSALTGSAPISAEMKSWVEDLLDMHLLEGYG STEAGAVFIDGQIQRPPVIDYKLVDVPDLGYFATDRPYPRGELLVKSEQMFPGYYKRPEITAEMFDEDG YYRTGDIVAELGPDHLEYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQTYVYGNSARSYLLAVVVP TEEALSRWDGDELKSRISDSLQDAARAAGLQSYEIPRDFLVETTPFTLENGLLTGIRKLARPKLKAHYG ERLEQLYTDLAEGQANELRELRRNGADRPVVETVSRAAVALLGASVTDLRSDAHFTDLGGDSLSALSFS NLLHEIFDVDVPVGVIVSPATDLAGVAAYIEGELRGSKRPTYASVHGRDATEVRARDLALGKFIDAKTL SAAPGLPRSGTEIRTVLLTGATGFLGRYLALEWLERMDLVDGKVICLVRARSDDEARARLDATFDTGDA TLLEHYRALAADHLEVIAGDKGEADLGLDHDTWQRLADTVDLIVDPAALVNHVLPYSQMFGPNALGTAE LIRIALTTTIKPYVYVSTIGVGQGISPEAFVEDADIREISATRRVDDSYANGYGNSKWAGEVLLREAHD WCGLPVSVFRCDMILADTTYSGQLNLPDMFTRLMLSLVATGIAPGSFYELDADGNRQRAHYDGLPVEFI AEAISTIGSQVTDGFETFHVMNPYDDGIGLDEYVDWLIEAGYPVHRVDDYATWLSRFETALRALPERQR QASLLPLLHNYQQPSPPVCGAMAPTDRFRAAVQDAKIGPDKDIPHVTADVIVKYISNLQMLGLL* 34 FadD9 MSINDQRLTRRVEDLYASDAQFAAASPNEAITQAIDQPGVALPQLIRMVMEGYADRPALGQRALRFVTD PDSGRTMVELLPRFETITYRELWARAGTLATALSAEPAIRPGDRVCVLGFNSVDYTTIDIALIRLGAVS VPLQTSAPVTGLRPIVTETEPTMIATSIDNLGDAVEVLAGHAPARLVVFDYHGKVDTHREAVEAARARL AGSVTIDTLAELIERGRALPATPIADSADDALALLIYTSGSTGAPKGAMYRESQVMSFWRKSSGWFEPS GYPSITLNFMPMSHVGGRQVLYGTLSNGGTAYFVAKSDLSTLFEDLALVRPTELCFVPRIWDMVFAEFH SEVDRRLVDGADRAALEAQVKAELRENVLGGRFVMALTGSAPISAEMTAWVESLLADVHLVEGYGSTEA GMVLNDGMVRRPAVIDYKLVDVPELGYFGTDQPYPRGELLVKTQTMFPGYYQRPDVTAEVFDPDGFYRT GDIMAKVGPDQFVYLDRRNNVLKLSQGEFIAVSKLEAVFGDSPLVRQIFIYGNSARAYPLAVVVPSGDA LSRHGIENLKPVISESLQEVARAAGLQSYEIPRDFIIETTPFTLENGLLTGIRKLARPQLKKFYGERLE RLYTELADSQSNELRELRQSGPDAPVLPTLCRAAAALLGSTAADVRPDAHFADLGGDSLSALSLANLLH EIFGVDVPVGVIVSPASDLRALADHIEAARTGVRRPSFASIHGRSATEVHASDLTLDKFIDAATLAAAP NLPAPSAQVRTVLLTGATGFLGRYLALEWLDRMDLVNGKLICLVRARSDEEAQARLDATFDSGDPYLVR HYRELGAGRLEVLAGDKGEADLGLDRVTWQRLADTVDLIVDPAALVNHVLPYSQLFGPNAAGTAELLRL ALTGKRKPYIYTSTIAVGEQIPPEAFTEDADIRAISPTRRIDDSYANGYANSKWAGEVLLREAHEQCGL PVTVFRCDMILADTSYTGQLNLPDMFTRLMLSLAATGIAPGSFYELDAHGNRQRAHYDGLPVEFVAEAI CTLGTHSPDRFVTYHVMNPYDDGIGLDEFVDWLNSPTSGSGCTIQRIADYGEWLQRFETSLRALPDRQR HASLLPLLHNYREPAKPICGSIAPTDQFRAAVQEAKIGPDKDIPHLTAAIIAKYISNLRLLGLL* 35 Synechococcus atgccgcagc ttgaagccag ccttgaactg gactttcaaa gcgagtccta caaagacgct elongatus PCC7942  60 YP.sub.--400610 tacagccgca tcaacgcgat cgtgattgaa ggcgaacaag aggcgttcga caactacaat (Synpcc7942.sub.-- 120 1593)(decarbonylase cgccttgctg agatgctgcc cgaccagcgg gatgagcttc acaagctagc caagatggaa DNA) 180 cagcgccaca tgaaaggctt tatggcctgt ggcaaaaatc tctccgtcac tcctgacatg 240 ggttttgccc agaaattttt cgagcgcttg cacgagaact tcaaagcggc ggctgcggaa 300 ggcaaggtcg tcacctgcct actgattcaa tcgctaatca tcgagtgctt tgcgatcgcg 360 gcttacaaca tctacatccc agtggcggat gcttttgccc gcaaaatcac ggagggggtc 420 gtgcgcgacg aatacctgca ccgcaacttc ggtgaagagt ggctgaaggc gaattttgat 480 gcttccaaag ccgaactgga agaagccaat cgtcagaacc tgcccttggt ttggctaatg 540 ctcaacgaag tggccgatga tgctcgcgaa ctcgggatgg agcgtgagtc gctcgtcgag 600 gactttatga ttgcctacgg tgaagctctg gaaaacatcg gcttcacaac gcgcgaaatc 660 atgcgtatgt ccgcctatgg ccttgcggcc gtttga 696 36 Synechococcus Met Pro Gln Leu Glu Ala Ser Leu Glu Leu Asp Phe Gln Ser Glu Ser elongatus PCC7942 1               5                   10                  15 YP.sub.--400610 Tyr Lys Asp Ala Tyr Ser Arg Ile Asn Ala Ile Val Ile Glu Gly Glu (Synpcc7942.sub.--             20                  25                  30 1593)(decarbonylase Gln Glu Ala Phe Asp Asn Tyr Asn Arg Leu Ala Glu Met Leu Pro Asp polypeptide)         35                  40                  45 Gln Arg Asp Glu Leu His Lys Leu Ala Lys Met Glu Gln Arg His Met     50                  55                  60 Lys Gly Phe Met Ala Cys Gly Lys Asn Leu Ser Val Thr Pro Asp Met 65                  70                  75                  80 Gly Phe Ala Gln Lys Phe Phe Glu Arg Leu His Glu Asn Phe Lys Ala                 85                  90                  95 Ala Ala Ala Glu Gly Lys Val Val Thr Cys Leu Leu Ile Gln Ser Leu             100                 105                 110 Ile Ile Glu Cys Phe Ala Ile Ala Ala Tyr Asn Ile Tyr Ile Pro Val         115                 120                 125 Ala Asp Ala Phe Ala Arg Lys Ile Thr Glu Gly Val Val Arg Asp Glu     130                 135                 140 Tyr Leu His Arg Asn Phe Gly Glu Glu Trp Leu Lys Ala Asn Phe Asp 145                 150                 155                 160 Ala Ser Lys Ala Glu Leu Glu Glu Ala Asn Arg Gln Asn Leu Pro Leu                 165                 170                 175 Val Trp Leu Met Leu Asn Glu Val Ala Asp Asp Ala Arg Glu Leu Gly             180                 185                 190 Met Glu Arg Glu Ser Leu Val Glu Asp Phe Met Ile Ala Tyr Gly Glu         195                 200                 205 Ala Leu Glu Asn Ile Gly Phe Thr Thr Arg Glu Ile Met Arg Met Ser     210                 215                 220 Ala Tyr Gly Leu Ala Ala Val 225                 230 37 Synecho- atgttcggtc ttatcggtca tctcaccagt ttggagcagg cccgcgacgt ttctcgcagg coccus elongatus   60 PCC7942 YP_400611 atgggctacg acgaatacgc cgatcaagga ttggagtttt ggagtagcgc tcctcctcaa (Synpcc7942_1594)  120 (AAR DNA) atcgttgatg aaatcacagt caccagtgcc acaggcaagg tgattcacgg tcgctacatc  180 gaatcgtgtt tcttgccgga aatgctggcg gcgcgccgct tcaaaacagc cacgcgcaaa  240 gttctcaatg ccatgtccca tgcccaaaaa cacggcatcg acatctcggc cttggggggc  300 tttacctcga ttattttcga gaatttcgat ttggccagtt tgcggcaagt gcgcgacact  360 accttggagt ttgaacggtt caccaccggc aatactcaca cggcctacgt aatctgtaga  420 caggtggaag ccgctgctaa aacgctgggc atcgacatta cccaagcgac agtagcggtt  480 gtcggcgcga ctggcgatat cggtagcgct gtctgccgct ggctcgacct caaactgggt  540 gtcggtgatt tgatcctgac ggcgcgcaat caggagcgtt tggataacct gcaggctgaa  600 ctcggccggg gcaagattct gcccttggaa gccgctctgc cggaagctga ctttatcgtg  660 tgggtcgcca gtatgcctca gggcgtagtg atcgacccag caaccctgaa gcaaccctgc  720 gtcctaatcg acgggggcta ccccaaaaac ttgggcagca aagtccaagg tgagggcatc  780 tatgtcctca atggcggggt agttgaacat tgcttcgaca tcgactggca gatcatgtcc  840 gctgcagaga tggcgcggcc cgagcgccag atgtttgcct gctttgccga ggcgatgctc  900 ttggaatttg aaggctggca tactaacttc tcctggggcc gcaaccaaat cacgatcgag  960 aagatggaag cgatcggtga ggcatcggtg cgccacggct tccaaccctt ggcattggca 1020 atttga 38 Synecho- Met Phe Gly Leu Ile Gly His Leu Thr Ser Leu Glu Gln Ala Arg Asp coccus elongatus 1               5                   10                  15 PCC7942 YP_400611 Val Ser Arg Arg Met Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu Glu (Synpcc7942_1594)             20                  25                  30 (AAR polypeptide) Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val Thr         35                  40                  45 Ser Ala Thr Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Cys Phe     50                  55                  60 Leu Pro Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg Lys 65                  70                  75                  80 Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile Ser                 85                  90                  95 Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu Ala             100                 105                 110 Ser Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu Arg Phe Thr         115                 120                 125 Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys Arg Gln Val Glu Ala     130                 135                 140 Ala Ala Lys Thr Leu Gly Ile Asp Ile Thr Gln Ala Thr Val Ala Val 145                 150                 155                 160 Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Asp                 165                 170                 175 Leu Lys Leu Gly Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln Glu             180                 185                 190 Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu Pro         195                 200                 205 Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala Ser     210                 215                 220 Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln Pro Cys 225                 230                 235                 240 Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu Gly Ser Lys Val Gln                 245                 250                 255 Gly Glu Gly Ile Tyr Val Leu Asn Gly Gly Val Val Glu His Cys Phe             260                 265                 270 Asp Ile Asp Trp Gln Ile Met Ser Ala Ala Glu Met Ala Arg Pro Glu         275                 280                 285 Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe Glu     290                 295                 300 Gly Trp His Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile Glu 305                 310                 315                 320 Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln Pro                 325                 330                 335 Leu Ala Leu Ala Ile             340 39 Prochlorococcus atgcaaacac tcgaatctaa taaaaaaact aatctagaaa attctattga tttacccgat mariunus CCMP1986  60 PMM0532 tttactactg attcttacaa agacgcttat agcaggataa atgcaatagt tattgaaggt (decarbonylase DNA) 120 gaacaagagg ctcatgataa ttacatttcc ttagcaacat taattcctaa cgaattagaa 180 gagttaacta aattagcgaa aatggagctt aagcacaaaa gaggctttac tgcatgtgga 240 agaaatctag gtgttcaagc tgacatgatt tttgctaaag aattcttttc caaattacat 300 ggtaattttc aggttgcgtt atctaatggc aagacaacta catgcctatt aatacaggca 360 attttaattg aagcttttgc tatatccgcg tatcacgttt acataagagt tgctgatcct 420 ttcgcgaaaa aaattaccca aggtgttgtt aaagatgaat atcttcattt aaattatgga 480 caagaatggc taaaagaaaa tttagcgact tgtaaagatg agctaatgga agcaaataag 540 gttaaccttc cattaatcaa gaagatgtta gatcaagtct cggaagatgc ttcagtacta 600 gctatggata gggaagaatt aatggaagaa ttcatgattg cctatcagga cactctcctt 660 gaaataggtt tagataatag agaaattgca agaatggcaa tggctgctat agtttaa 717 40 Prochlorococcus Met Gln Thr Leu Glu Ser Asn Lys Lys Thr Asn Leu Glu Asn Ser Ile mariunus CCMP1986 1               5                   10                  15 PMM0532 Asp Leu Pro Asp Phe Thr Thr Asp Ser Tyr Lys Asp Ala Tyr Ser Arg (decarbonylase             20                  25                  30 polypeptide) Ile Asn Ala Ile Val Ile Glu Gly Glu Gln Glu Ala His Asp Asn Tyr         35                  40                  45 Ile Ser Leu Ala Thr Leu Ile Pro Asn Glu Leu Glu Glu Leu Thr Lys     50                  55                  60 Leu Ala Lys Met Glu Leu Lys His Lys Arg Gly Phe Thr Ala Cys Gly 65                  70                  75                  80 Arg Asn Leu Gly Val Gln Ala Asp Met Ile Phe Ala Lys Glu Phe Phe                 85                  90                  95 Ser Lys Leu His Gly Asn Phe Gln Val Ala Leu Ser Asn Gly Lys Thr             100                 105                 110 Thr Thr Cys Leu Leu Ile Gln Ala Ile Leu Ile Glu Ala Phe Ala Ile         115                 120                 125 Ser Ala Tyr His Val Tyr Ile Arg Val Ala Asp Pro Phe Ala Lys Lys     130                 135                 140 Ile Thr Gln Gly Val Val Lys Asp Glu Tyr Leu His Leu Asn Tyr Gly 145                 150                 155                 160 Gln Glu Trp Leu Lys Glu Asn Leu Ala Thr Cys Lys Asp Glu Leu Met                 165                 170                 175 Glu Ala Asn Lys Val Asn Leu Pro Leu Ile Lys Lys Met Leu Asp Gln             180                 185                 190 Val Ser Glu Asp Ala Ser Val Leu Ala Met Asp Arg Glu Glu Leu Met         195                 200                 205 Glu Glu Phe Met Ile Ala Tyr Gln Asp Thr Leu Leu Glu Ile Gly Leu     210                 215                 220 Asp Asn Arg Glu Ile Ala Arg Met Ala Met Ala Ala Ile Val 41 Prochlorococcus atgtttgggc ttataggtca ttcaactagt tttgaagatg caaaaagaaa ggcttcatta marinu   60 CCMP1986 PMM0533 ttgggctttg atcatattgc ggatggtgat ttagatgttt ggtgcacagc tccacctcaa (NP_892651)(DNA)  120 ctagttgaaa atgtagaggt taaaagtgct ataggtatat caattgaagg ttcttatatt  180 gattcatgtt tcgttcctga aatgctttca agatttaaaa cggcaagaag aaaagtatta  240 aatgcaatgg aattagctca aaaaaaaggt attaatatta ccgctttggg ggggttcact  300 tctatcatct ttgaaaattt taatctcctt caacataagc agattagaaa cacttcacta  360 gagtgggaaa ggtttacaac tggtaatact catactgcgt gggttatttg caggcaatta  420 gagatgaatg ctcctaaaat aggtattgat cttaaaagcg caacagttgc tgtagttggt  480 gctactggag atataggcag tgctgtttgt cgatggttaa tcaataaaac aggtattggg  540 gaacttcttt tggtagctag gcaaaaggaa cccttggatt ctttgcaaaa ggaattagat  600 ggtggaacta tcaaaaatct agatgaagca ttgcctgaag cagatattgt tgtatgggta  660 gcaagtatgc caaagacaat ggaaatcgat gctaataatc ttaaacaacc atgtttaatg  720 attgatggag gttatccaaa gaatctagat gaaaaatttc aaggaaataa tatacatgtt  780 gtaaaaggag gtatagtaag attcttcaat gatataggtt ggaatatgat ggaactagct  840 gaaatgcaaa atccccagag agaaatgttt gcatgctttg cagaagcaat gattttagaa  900 tttgaaaaat gtcatacaaa ctttagctgg ggaagaaata atatatctct cgagaaaatg  960 gagtttattg gagctgcttc tgtaaagcat ggcttctctg caattggcct agataagcat 1020 ccaaaagtac tagcagtttg a 1041 42 Prochloro- Met Phe Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys Arg coccus marinu 1               5                   10                  15 CCMP1986 PMM0533 Lys Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu Asp (NP_892651)             20                  25                  30 (polypeptide) Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val Lys         35                  40                  45 Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys Phe     50                  55                  60 Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val Leu 65                  70                  75                  80 Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala Leu                 85                  90                  95 Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln His             100                 105                 110 Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe Thr Thr Gly         115                 120                 125 Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln Leu Glu Met Asn Ala     130                 135                 140 Pro Lys Ile Gly Ile Asp Leu Lys Ser Ala Thr Val Ala Val Val Gly 145                 150                 155                 160 Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile Asn Lys                 165                 170                 175 Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro Leu             180                 185                 190 Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu Asp         195                 200                 205 Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met Pro     210                 215                 220 Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys Leu Met 225                 230                 235                 240 Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu Lys Phe Gln Gly Asn                 245                 250                 255 Asn Ile His Val Val Lys Gly Gly Ile Val Arg Phe Phe Asn Asp Ile             260                 265                 270 Gly Trp Asn Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg Glu         275                 280                 285 Met Phe Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys Cys     290                 295                 300 His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys Met 305                 310                 315                 320 Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile Gly                 325                 330                 335 Leu Asp Lys His Pro Lys Val Leu Ala Val             340                 345 43 Nostoc punctiforme PCC ATGAGCCAAACGGAACTTTTTGAAAAGGTCAAGAAAATCGTCATCGAACAACTGAGTGTTGAAGATGCTT 73102_acp Accession# CCAAAATCACTCCACAAGCTAAGTTTATGGAAGATTTAGGAGCTGATTCCCTGGATACTGTTGAACTCGT YP_001867863 (DNA) GATGGCTTTGGAAGAAGAATTTGATATCGAAATTCCCGACGAAGCTGCCGAGCAGATTGTATCGGTTCAA GACGCAGTAGATTACATCAATAACAAAGTTGCTGCATCAGCTTAA 44 Nostoc punctiforme PCC MSQTELFEKVKKIVIEQLSVEDASKITPQAKFMEDLGADSLDTVELVMALEEEFDIEIPDEAAEQIVSVQ 73102_acp Accession# DAVDYINNKVAASA YP_001867863 (polypeptide) 45 Synechocystis sp. PCC ATGAATCAGGAAATTTTTGAAAAAGTAAAAAAAATCGTCGTGGAACAGTTGGAAGTGGATCCTGACAAAG 6803_acp Accession # TGACCCCCGATGCCACCTTTGCCGAAGATTTAGGGGCTGATTCCCTCGATACAGTGGAATTGGTCATGGC NP_440632.1 (DNA) CCTGGAAGAAGAGTTTGATATTGAAATTCCCGATGAAGTGGCGGAAACCATTGATACCGTGGGCAAAGCC GTTGAGCATATCGAAAGTAAATAA 46 Synechocystis sp. PCC MNQEIFEKVKKIVVEQLEVDPDKVTPDATFAEDLGADSLDTVEL 6803_acp Accession # VMALEEEFDIEIPDEVAETIDTVGKAVEHIESK NP_440632.1 (polypeptide) 47 Prochlorococcus ATGTCACAAGAAGAAATCCTTCAAAAAGTATGCTCTATTGTTTCTGAGCAACTAAGTGTTGAATCAGCCG marinu; AAGTAAAATCTGATTCAAACTTTCAAAATGATTTAGGTGCAGACTCCCTAGACACCGTAGAGCTAGTTAT subsp. pastoris str. GGCTCTTGAAGAAGCATTTGATATCGAGATACCTGATGAAGCAGCTGAAGGTATCGCAACAGTAGGAGAT CCMP1986_acp GCTGTTAAATTCATCGAAGAAAAAAAAGGTTAA Accession# NP_893725. (DNA) 48 Prochlorococcus MSQEEILQKVCSIVSEQLSVESAEVKSDSNFQNDLGADSLDTVELVMALEEAFDIEIPDEAAEGIATVGD marinu; AVKFIEEKKG subsp. pastoris str. CCMP1986_acp Accession# NP_893725. (polypeptide) 49 Synechococcus elongatu ATGAGCCAAGAAGACATCTTCAGCAAAGTCAAAGACATTGTGGCTGAGCAGCTGAGTGTGGATGTGGCTG PCC 7942_acp AAGTCAAGCCAGAATCCAGCTTCCAAAACGATCTGGGAGCGGACTCGCTGGACACCGTGGAACTGGTGAT Accession# YP_399555 GGCTCTGGAAGAGGCTTTCGATATCGAAATCCCCGATGAAGCCGCTGAAGGCATTGCGACCGTTCAAGAC (DNA) GCCGTCGATTTCATCGCTAGCAAAGCTGCCTAG 50 Synechococcus elongatu MSQEDIFSKVKDIVAEQLSVDVAEVKPESSFQNDLGADSLDTVELVMALEEAFDIEIPDEAAEGIATVQD PCC 7942_acp AVDFIASKAA Accession# YP_399555 (polypeptide) 51 Nostoc sp. PCC ATGAGCCAATCAGAAACTTTTGAAAAAGTCAAAAAAATTGTTATCGAACAACTAAGTGTGGAGAACCCTG 7120_acp Accession# ACACAGTAACTCCAGAAGCTAGTTTTGCCAACGATTTACAGGCTGATTCCCTCGATACAGTAGAACTAGT NP_487382.1 (DNA) AATGGCTTTGGAAGAAGAATTTGATATCGAAATTCCCGATGAAGCCGCAGAGAAAATTACCACTGTTCAA GAAGCGGTGGATTACATCAATAACCAAGTTGCCGCATCAGCTTAA 52 Nostoc sp. PCC MSQSETFEKVKKIVIEQLSVENPDTVTPEASFANDLQADSLDTVELVMALEEEFDIEIPDEAAEKITTVQ 7120_acp Accession# EAVDYINNQV NP_487382.1 AASA (polypeptide) 53 B.subtilis sfp ATGAAGATTTACGGAATTTATATGGACCGCCCGCTTTCACAGGAAGAAAATGAACGGTTCATGACTTTCA (synthesized)as in TATCACCTGAAAAACGGGAGAAATGCCGGAGATTTTATCATAAAGAAGATGCTCACCGCACCCTGCTGGG accession# X63158.1 AGATGTGCTCGTTCGCTCAGTCATAAGCAGGCAGTATCAGTTGGACAAATCCGATATCCGCTTTAGCACG (DNA) CAGGAATACGGGAAGCCGTGCATCCCTGATCTTCCCGACGCTCATTTCAACATTTCTCACTCCGGCCGCT GGGTCATTGGTGCGTTTGATTCACAGCCGATCGGCATAGATATCGAAAAAACGAAACCGATCAGCCTTGA GATCGCCAAGCGCTTCTTTTCAAAAACAGAGTACAGCGACCTTTTAGCAAAAGACAAGGACGAGCAGACA GACTATTTTTATCATCTATGGTCAATGAAAGAAAGCTTTATCAAACAGGAAGGCAAAGGCTTATCGCTTC CGCTTGATTCCTTTTCAGTGCGCCTGCATCAGGACGGACAAGTATCCATTGAGCTTCCGGACAGCCATTC CCCATGCTATATCAAAACGTATGAGGTCGATCCCGGCTACAAAATGGCTGTATGCGCCGCACACCCTGAT TTCCCCGAGGATATCACAATGGTCTCGTACGAAGAGCTTTTATAA 54 B.subtilis sfp MKIYGIYMDRPLSQEENERFMTFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQYQLDKSDIRFSTC (synthesized)as EYGKPCIPD in accession# LPDAHFNISHSGRWVIGAFDSQPIGIDIEKTKPISLEIAKRFFSKTEYSDLLAKDKDEQTDYFYHLWSMKE X63158.1 SFIKQEGKG (polypeptide) LSLPLDSFSVRLHQDGQVSIELPDSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELL 55 birA from 1 ttgggcgtgt cgcccttaaa gcgcgctttt cgacgcgacc ccactacatt ggcttccatg Corynebacterium 61 aacgttgaca tttcacgatc cagagagccg ctaaacgttg agctcctgaa ggaaaaattg glutamicum 121 ctccaaaacg gtgactttgg ccaggtcatt tacgaaaaag tgacaggctc cactaatgct (YP_224991), DNA 181 gacttgctgg cacttgcagg ttctggcgct ccaaactgga cggtgaaaac tgtcgagttt 241 caagatcatg cgcgtgggcg actcggccgc ccgtggtctg cccctgaggg ttcccaaaca 301 atcgtgtctg tgctcgttca actatctatt gatcaagtgg accggattgg cactattcca 361 ctcgcggcgg gactcgctgt catggatgcg ttgaatgacc tcggtgtgga aggtgccgga 421 ctgaaatggc ccaacgatgt tcaaatccac ggcaagaaac tctgcggcat cctggtggaa 481 gccaccggct ttgattccac cccaacagtt gtcatcggtt ggggcactaa tatcagcctg 541 actaaagagg agcttcctgt tcctcatgca acttccctcg cattggaagg tgttgaagtc 601 gacagaacca cattccttat taatatgctc acacatctgc atactcgact ggaccagtgg 661 cagggtccaa gtgtggattg gctcgatgat taccgtgcgg tatgttccag tattggccaa 721 gatgttcgag tgcttctacc tggggataaa gaactcttag gtgaagcgat cggtgtcgcg 781 actggcggag aaattcgtgt tcgcgatgct tcgggcaccg ttcacaccct caacgccggt 841 gaaattacgc accttcgcct gcagtaa 56 birA from 1 ttgggcgtgt cgcccttaaa gcgcgctttt cgacgcgacc ccactacatt ggcttccatg birA from 61 aacgttgaca tttcacgatc cagagagccg ctaaacgttg agctcctgaa ggaaaaattg Corynebacterium 121 ctccaaaacg gtgactttgg ccaggtcatt tacgaaaaag tgacaggctc cactaatgct glutamicum 181 gacttgctgg cacttgcagg ttctggcgct ccaaactgga cggtgaaaac tgtcgagttt (YP_224991), 241 caagatcatg cgcgtgggcg actcggccgc ccgtggtctg cccctgaggg ttcccaaaca synthetic DNA 301 atcgtgtctg tgctcgttca actatctatt gatcaagtgg accggattgg cactattcca 361 ctcgcggcgg gactcgctgt catggatgcg ttgaatgacc tcggtgtgga aggtgccgga 421 ctgaaatggc ccaacgatgt tcaaatccac ggcaagaaac tctgcggcat cctggtggaa 481 gccaccggct ttgattccac cccaacagtt gtcatcggtt ggggcactaa tatcagcctg 541 actaaagagg agcttcctgt tcctcatgca acttccctcg cattggaagg tgttgaagtc 601 gacagaacca cattccttat taatatgctc acacatctgc atactcgact ggaccagtgg 661 cagggtccaa gtgtggattg gctcgatgat taccgtgcgg tatgttccag tattggccaa 721 gatgttcgag tgcttctacc tggggataaa gaactcttag gtgaagcgat cggtgtcgcg 781 actggcggag aaattcgtgt tcgcgatgct tcgggcaccg ttcacaccct caacgccggt 841 gaaattacgc accttcgcct gcagtaa 57 Corynebacterium 1 MNVDISRSRE PLNVELLKEK LLQNGDFGQV IYEKVTGSTN ADLLALAGSG APNWTVKTVE glutamicum 61 FQDHARGRLG RPWSAPEGSQ TIVSVLVQLS IDQVDRIGTI PLAAGLAVMD ALNDLGVEGA (YP_224991), 121 GLKWPNDVQI HGKKLCGILV EATGFDSTPT VVIGWGTNIS LTKEELPVPH ATSLALEGVE Protein 181 VDRTTFLINM LTHLHTRLDQ WQGPSVDWLD DYRAVCSSIG QDVRVLLPGD KELLGEAIGV 241 ATGGEIRVRD ASGTVHTLNA GEITHLRLQ. 58 accDA1 (dtsR) from 1 ATGACCATTT CCTCACCTTT GATTGACGTC GCCAACCTTC CAGACATCAA CACCACTGCC Corynebacterium 61 GGCAAGATCG CCGACCTTAA GGCTCGCCGC GCGGAAGCCC ATTTCCCCAT GGGTGAAAAG glutamicum 121 GCAGTAGAGA AGGTCCACGC TGCTGGACGC CTCACTGCCC GTGAGCGCTT GGATTACTTA (YP_224991), DNA 181 CTCGATGAGG GCTCCTTCAT CGAGACCGAT CAGCTGGCTC GCCACCGCAC CACCGCTTTC 241 GGCCTGGGCG CTAAGCGTCC TGCAACCGAC GGCATCGTGA CCGGCTGGGG CACCATTGAT 301 GGACGCGAAG TCTGCATCTT CTCGCAGGAC GGCACCGTAT TCGGTGGCGC GCTTGGTGAG 361 GTGTACGGCG AAAAGATGAT CAAGATCATG GAGCTGGCAA TCGACACCGG CCGCCCATTG 421 ATCGGTCTTT ACGAAGGCGC TGGCGCTCGT ATTCAGGACG GCGCTGTCTC CCTGGACTTC 481 ATTTCCCAGA CCTTCTACCA AAACATTCAG GCTTCTGGCG TTATCCCACA GATCTCCGTC 541 ATCATGGGCG CATGTGCAGG TGGCAACGCT TACGGCGAAG CTCTGACCGA CTTCGTGGTC 601 ATGGTGGACA AGACCTCCAA GATGTTCGTT ACCGGCCCAG ACGTGATCAA GACCGTCACC 661 GGCGAGGAAA TCACCCAGGA AGAGCTTGGC GGAGCAACCA CCCACATGGT GACCGCTGGT 721 AACTCCCACT ACACCGCTGC GACCGATGAG GAAGCACTGG ATTGGGTACA GGACCTGGTG 781 TCCTTCCTCC CATCCAACAA TCGCTCCTAC GCACCGATGG AAGACTTCGA CGAGGAAGAA 841 GGCGGCGTTG AAGAAAACAT CACCGCTGAC GATCTGAAGC TCGACGAGAT CATCCCAGAT 901 TCCGCGACCG TTCCTTACGA CGTCCGCGAT GTCATCGAAT GCCTCACCGA CGATGGCGAA 961 TACCTGGAAA TCCAGGCAGA CCGCGCAGAA AACGTTGTTA TTGCATTCGG CCGCATCGAA 1021 GGCCAGTCCG TTGGCTTTGT TGCCAACCAG CCAACCCAGT TCGCTGGCTG CCTGGACATC 1081 GACTCCTCTG AGAAGGCAGC TCGCTTCGTC CGCACCTGCG ACGCGTTCAA CATCCCAATC 1141 GTCATGCTTG TCGACGTCCC CGGCTTCCTC CCAGGCGCAG GCCAGGAGTA CGGTGGCATT 1201 CTGCGTCGTG GCGCAAAGCT GCTCTACGCA TACGGCGAAG CAACCGTTCC AAAGATCACC 1261 GTCACCATGC GTAAGGCTTA CGGCGGAGCG TACTGCGTGA TGGGTTCCAA GGGCTTGGGC 1321 TCTGACATCA ACCTTGCATG GCCAACCGCA CAGATCGCCG TCATGGGCGC TGCTGGCGCA 1381 GTTGGATTCA TCTACCGCAA GGAGCTCATG GCAGCTGATG CCAAGGGCCT CGATACCGTA 1441 GCTCTGGCTA AGTCCTTCGA GCGCGAGTAT GAAGACCACA TGCTCAACCC GTACCACGCT 1501 GCAGAACGTG GCCTGATCGA CGCCGTGATC CTGCCAAGCG AAACCCGCGG ACAGATTTCC 1561 CGCAACCTTC GCCTGCTCAA GCACAAGAAC GTCACTCGCC CTGCTCGCAA GCACGGCAAC 1621 ATGCCACTGT AA 59 accDA1 (dtsR) 1 MTISSPLIDV ANLPDINTTA GKIADLKARR AEAHFPMGEK AVEKVHAAGR LTARERLDYL from 61 LDEGSFIETD QLARHRTTAF GLGAKRPATD GIVTGWGTID GREVCIFSQD GTVFGGALGE Corynebacterium 121 VYGEKMIKIM ELAIDTGRPL IGLYEGAGAR IQDGAVSLDF ISQTFYQNIQ ASGVIPQISV glutamicum 181 IMGACAGGNA YGPALTDFVV MVDKTSKMFV TGPDVIKTVT GEEITQEELG GATTHMVTAG (YP_224991), 241 NSHYTAATDE EALDWVQDLV SFLPSNNRSY APMEDFDEEE GGVEENITAD DLKLDEIIPD Protein 301 SATVPYDVRD VIECLTDDGE YLEIQADRAE NVVIAFGRIE GQSVGFVANQ PTQFAGCLDI 361 DSSEKAARFV RTCDAFNIPI VMLVDVPGFL PGAGQEYGGI LRRGAKLLYA YGEATVPKIT 421 VTMRKAYGGA YCVMGSKGLG SDINLAWPTA QIAVMGAAGA VGFIYRKELM AADAKGLDTV 481 ALAKSFEREY EDHMLNPYHA AERGLIDAVI LPSETRGQIS RNLRLLKHKN VTRPARKHGN 541 MPL 60 accCB from 1 atgtcagtcg agactcgcaa gatcaccaag gttcttgtcg ctaaccgtgg tgagattgca Corynebacterium 61 atccgcgtgt tccgtgcagc tcgagatgaa ggcatcggat ctgtcgccgt ctacgcagag glutamicum 121 ccagatgcag atgcaccatt cgtgtcatat gcagacgagg cttttgccct cggtggccaa (YP_224991, DNA 181 acatccgctg agtcctacct tgtcattgac aagatcatcg atgcggcccg caagtccggc 241 gccgacgcca tccaccccgg ctacggcttc ctcgcagaaa acgctgactt cgcagaagca 301 gtcatcaacg aaggcctgat ctggattgga ccttcacctg agtccatccg ctccctcggc 361 gacaaggtca ccgctcgcca catcgcagat accgccaagg ctccaatggc tcctggcacc 421 aaggaaccag taaaagacgc agcagaagtt gtggctttcg ctgaagaatt cggtctccca 481 atcgccatca aggcagcttt cggtggcggc ggacgtggca tgaaggttgc ctacaagatg 541 gaagaagtcg ctgacctctt cgagtccgca acccgtgaag caaccgcagc gttcggccgc 601 ggcgagtgct tcgtggagcg ctacctggac aaggcacgcc acgttgaggc tcaggtcatc 661 gccgataagc acggcaacgt tgttgtcgcc ggaacccgtg actgctccct gcagcgccgt 721 ttccagaagc tcgtcgaaga agcaccagca ccattcctca ccgatgacca gcgcgagcgt 781 ctccactcct ccgcgaaggc tatctgtaag gaagctggct actacggtgc aggcaccgtt 841 gagtacctcg ttggctccga cggcctgatc tccttcctcg aggtcaacac ccgcctccag 901 gtggaacacc cagtcaccga agagaccacc ggcatcgacc tggtccgcga aatgttccgc 961 atcgcagaag gccacgagct ctccatcaag gaagatccag ctccacgcgg ccacgcattc 1021 gagttccgca tcaacggcga agacgctggc tccaacttca tgcctgcacc aggcaagatc 1081 accagctacc gcgagccaca gggcccaggc gtccgcatgg actccggtgt cgttgaaggt 1141 tccgaaatct ccggacagtt cgactccatg ctggcaaagc tgatcgtttg gggcgacacc 1201 cgcgagcagg ctctccagcg ctcccgccgt gcacttgcag agtacgttgt cgagggcatg 1261 ccaaccgtta tcccattcca ccagcacatc gtggaaaacc cagcattcgt gggcaacgac 1321 gaaggcttcg agatctacac caagtggatc gaagaggttt gggataaccc aatcgcacct 1381 tacgttgacg cttccgagct cgacgaagat gaggacaaga ccccagcaca gaaggttgtt 1441 gtggagatca acggccgtcg cgttgaggtt gcactcccag gcgatctggc actcggtggc 1501 accgctggtc ctaagaagaa ggccaagaag cgtcgcgcag gtggtgcaaa ggctggcgta 1561 tccggcgatg cagtggcagc tccaatgcag ggcactgtca tcaaggtcaa cgtcgaagaa 1621 ggcgctgaag tcaacgaagg cgacaccgtt gttgtcctcg aggctatgaa gatggaaaac 1681 cctgtgaagg ctcataagtc cggaaccgta accggcctta ctgtcgctgc aggcgagggt 1741 gtcaacaagg gcgttgttct cctcgagatc aagtaa 61 accCB from 1 MSVETRKITK VLVANRGEIA IRVFRAARDE GIGSVAVYAE PDADAPFVSY ADEAFALGGQ Corynebacterium 61 TSAESYLVID KIIDAARKSG ADAIHPGYGF LAENADFAEA VINEGLIWIG PSPESIRSLG glutamicum 121 DKVTARHIAD TAKAPMAPGT KEPVKDAAEV VAFAEEFGLP IAIKAAFGGG GRGMKVAYKM (YP_224991, 181 EEVADLFESA TREATAAFGR GECFVERYLD KARHVEAQVI ADKHGNVVVA GTRDCSLQRR Protein 241 FQKLVEEAPA PFLTDDQRER LHSSAKAICK EAGYYGAGTV EYLVGSDGLI SFLEVNTRLQ 301 VEHPVTEETT GIDLVREMFR IAEGHELSIK EDPAPRGHAF EFRINGEDAG SNFMPAPGKI 361 TSYREPQGPG VRMDSGVVEG SEISGQFDSM LAKLIVWGDT REQALQRSRR ALAEYVVEGM 421 PTVIPFHQHI VENPAFVGND EGFEIYTKWI EEVWDNPIAP YVDASELDED EDKTPAQKVV 481 VEINGRRVEV ALPGDLALGG TAGPKKKAKK RRAGGAKAGV SGDAVAAPMQ GTVIKVNVEE 541 GAEVNEGDTV VVLEAMKMEN PVKAHKSGTV TGLTVAAGEG VNKGVVLLEI K 62 OP80 trc3 + FH Key Location/Qualifiers accDA1CB + birA FH (pAS119.50D) FT misc_feature 6622 . . . 6713 FT /note = “lacZalpha” FT /translation = ” “A.CGIFSLRICAVFHTAYGALSVQSALMPH FT misc_feature complement(7003 . . . 8013) FT /note = “aadA1- aminoglycoside 3′- adenylyltransferase” FT /translation = “MRSRNWSRTLTERSGGNGAVAVFMACYDCFFGVQSMPRASKQQA FT RYAVGRCLMLWSSNDVTQQGSRPKTKLNIMREAVIAEVSTQLSEVVGVIERHLEPTLL FT AVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVE FT VTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVAL FT VGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIA FT PKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVV FT GK” FT misc_feature complement(9046 . . . 9996) FT /note = “repA protein” FT /translation = “MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRK FT ERTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAK FT FSSEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHK FT ANIEISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTL FT IFQVELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTS FT FEAKFLSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI” FT vector join(1 . . . 329, 6619 . . . 10025) FT /source = “pCL1920revised” FT /type = “Custom cloned vector” FT insert 330 . . . 6621 FT /source = “pCL1920Ptrc” FT /type = “Custom cloned insert” FT misc_feature 6425 . . . 6582 FT /note = “TERM rrnB T1 and T2 transcriptional terminators” FT misc_feature 2037 . . . 2063 FT /note = “mini cistron ORF” FT misc_feature 2052 . . . 2057 FT /note = “RBS (Reinitiation)” FT misc_feature 1847 . . . 2036 FT /note = “Ptrc” FT misc_feature 1847 . . . 1852 FT /note = “−35 region” FT misc_feature 1870 . . . 1874 FT /note = “−10 region” FT misc_feature 1882 . . . 1902 FT /note = “lacO- lac operator” FT misc_feature 1918 . . . 1987 FT /note = “rrnB antitermination signal” FT misc_feature 2000 . . . 2008 FT /note = “g10 RBS (gene 10 region)” FT misc_feature 2023 . . . 2027 FT /note = “RBS” FT misc_feature 543 . . . 1625 FT /note = “Lac Repressor lacIq ORF” FT /translation = “VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE FT LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS FT GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FT FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG FT DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT FT EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT FT QTASPRALADSLMQLARQVSRLESGQ” FT modified_base 1399 . . . 1399 FT /note = “Change from C to T” FT modified_base 1644 . . . 1644 FT /note = “Change from G to A” FT misc_feature 1875 . . . 1875 FT /note = “trc3” FT gene 2067 . . . 3698 FT /note = “dtsR1(accDA1)” FT /translation = “MTISSPLIDVANLPDINTTAGKIADLKARRAEAHFPMGEKAVEK FT VHAAGRLTARERLDYLLDEGSFIETDQLARHRTTAFGLGAKRPATDGIVTGWGTIDGR FT EVCIFSQDGTVFGGALGEVYGEKMIKIMELAIDTGRPLIGLYEGAGARIQDGAVSLDF FT ISQTFYQNIQASGVIPQISVIMGACAGGNAYGPALTDFVVMVDKTSKMFVTGPDVIKT FT VTGEEITQEELGGATTHMVTAGNSHYTAATDEEALDWVQDLVSFLPSNNRSYAPMEDF FT DEEEGGVEENITADDLKLDEIIPDSATVPYDVRDVIECLTDDGEYLEIQADRAENVVI FT AFGRIEGQSVGFVANQPTQFAGCLDIDSSEKAARFVRTCDAFNIPIVMLVDVPGFLPG FT AGQEYGGILRRGAKLLYAYGEATVPKITVTMRKAYGGAYCVMGSKGLGSDINLAWPTA FT QIAVMGAAGAVGFIYRKELMAADAKGLDTVALAKSFEREYEDHMLNPYHAAERGLIDA FT VILPSETRGQISRNLRLLKHKNVTRPARKHGNMPL” FT gene 3712 . . . 5487 FT /note = “C. glutamicum accCB” FT /translation = “MSVETRKITKVLVANRGEIAIRVFRAARDEGIGSVAVYAEPDAD FT APFVSYADEAFALGGQTSAESYLVIDKIIDAARKSGADAIHPGYGFLAENADFAEAVI FT NEGLIWIGPSPESIRSLGDKVTARHIADTAKAPMAPGTKEPVKDAAEVVAFAEEFGLP FT IAIKAAFGGGGRGMKVAYKMEEVADLFESATREATAAFGRGECFVERYLDKARHVEAQ FT VIADKHGNVVVAGTRDCSLQRRFQKLVEEAPAPFLTDDQRERLHSSAKAICKEAGYYG FT AGTVEYLVGSDGLISFLEVNTRLQVEHPVTEETTGIDLVREMFRIAEGHELSIKEDPA FT PRGHAFEFRINGEDAGSNFMPAPGKITSYREPQGPGVRMDSGVVEGSEISGQFDSMLA FT KLIVWGDTREQALQRSRRALAEYVVEGMPTVIPFHQHIVENPAFVGNDEGFEIYTKWI FT EEVWDNPIAPYVDASELDEDEDKTPAQKVVVEINGRRVEVALPGDLALGGTAGPKKKA FT KKRRAGGAKAGVSGDAVAAPMQGTVIKVNVEEGAEVNEGDTVVVLEAMKMENPVKAHK FT SGTVTGLTVAAGEGVNKGVVLLEIK” FT misc_feature 3727 . . . 3729 FT /note = “rare Arg codon, change to  CGT or CGC” FT misc_feature 3712 . . . 3714 FT /note = “GTG start codon, change to ATG” FT gene 5507 . . . 6316 FT /note = “birA_Cg_opt” FT /translation = “MNVDISRSREPLNVELLKEKLLQNGDFGQVIYEKVTGSTNADLL FT ALAGSGAPNWTVKTVEFQDHARGRLGRPWSAPEGSQTIVSVLVQLSIDQVDRIGTIPL FT AAGLAVMDALNDLGVEGAGLKWPNDVQIHGKKLCGILVEATGFDSTPTVVIGWGTNIS FT LTKEELPVPHATSLALEGVEVDRTTFLINMLTHLHTRLDQWQGPSVDWLDDYRAVCSS FT IGQDVRVLLPGDKELLGEAIGVATGGEIRVRDASGTVHTLNAGEITHLRLQ” FT misc_feature 5493 . . . 5498 FT /note = “RBS” FT source 1 . . . 10025 FT /dnas_title = “OP80 trc3 + accDA1CB + birA” SQ 10025 BP; 2326 A; 2661 C; 2606 G; 2432 T; CACTATACCA ATTGAGATGG GCTAGTCAAT GATAATTACT AGTCCTTTTC CTTTGAGTTG 60 TGGGTATCTG TAAATTCTGC TAGACCTTTG CTGGAAAACT TGTAAATTCT GCTAGACCCT 120 CTGTAAATTC CGCTAGACCT TTGTGTGTTT TTTTTGTTTA TATTCAAGTG GTTATAATTT 180 ATAGAATAAA GAAAGAATAA AAAAAGATAA AAAGAATAGA TCCCAGCCCT GTGTATAACT 240 CACTACTTTA GTCAGTTCCG CAGTATTACA AAAGGATGTC GCAAACGCTG TTTGCTCCTC 300 TACAAAACAG ACCTTAAAAC CCTAAAGGCg tcGGCATCCG CTTACAGACA AGCTGTGACC 360 GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG CGCGAGGCAG 420 CAGATCAATT CGCGCGCGAA GGCGAAGCGG CATGCATTTA CGTTGACACC ATCGAATGGT 480 GCAAAACCTT TCGCGGTATG GCATGATAGC GCCCGGAAGA GAGTCAATTC AGGGTGGTGA 540 ATGTGAAACC AGTAACGTTA TACGATGTCG CAGAGTATGC CGGTGTCTCT TATCAGACCG 600 TTTCCCGCGT GGTGAACCAG GCCAGCCACG TTTCTGCGAA AACGCGGGAA AAAGTGGAAG 660 CGGCGATGGC GGAGCTGAAT TACATTCCCA ACCGCGTGGC ACAACAACTG GCGGGCAAAC 720 AGTCGTTGCT GATTGGCGTT GCCACCTCCA GTCTGGCCCT GCACGCGCCG TCGCAAATTG 780 TCGCGGCGAT TAAATCTCGC GCCGATCAAC TGGGTGCCAG CGTGGTGGTG TCGATGGTAG 840 AACGAAGCGG CGTCGAAGCC TGTAAAGCGG CGGTGCACAA TCTTCTCGCG CAACGCGTCA 900 GTGGGCTGAT CATTAACTAT CCGCTGGATG ACCAGGATGC CATTGCTGTG GAAGCTGCCT 960 GCACTAATGT TCCGGCGTTA TTTCTTGATG TCTCTGACCA GACACCCATC AACAGTATTA 1020  TTTTCTCCCA TGAAGACGGT ACGCGACTGG GCGTGGAGCA TCTGGTCGCA TTGGGTCACC 1080  AGCAAATCGC GCTGTTAGCG GGCCCATTAA GTTCTGTCTC GGCGCGTCTG CGTCTGGCTG 1140 GCTGGCATAA ATATCTCACT CGCAATCAAA TTCAGCCGAT AGCGGAACGG GAAGGCGACT 1200  GGAGTGCCAT GTCCGGTTTT CAACAAACCA TGCAAATGCT GAATGAGGGC ATCGTTCCCA 1260  CTGCGATGCT GGTTGCCAAC GATCAGATGG CGCTGGGCGC AATGCGCGCC ATTACCGAGT 1320  CCGGGCTGCG CGTTGGTGCG GATATCTCGG TAGTGGGATA CGACGATACC GAAGACAGCT 1380  CATGTTATAT CCCGCCGTtA ACCACCATCA AACAGGATTT TCGCCTGCTG GGGCAAACCA 1440  GCGTGGACCG CTTGCTGCAA CTCTCTCAGG GCCAGGCGGT GAAGGGCAAT CAGCTGTTGC 1500  CCGTCTCACT GGTGAAAAGA AAAACCACCC TGGCGCCCAA TACGCAAACC GCCTCTCCCC 1560  GCGCGTTGGC CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC 1620 AGTGAGCGCA ACGCAATTAA TGTaAGTTAG CGCGAATTGA TCTGGTTTGA CAGCTTATCA 1680  TCGACTGCAC GGTGCACCAA TGCTTCTGGC GTCAGGCAGC CATCGGAAGC TGTGGTATGG 1740 CTGTGCAGGT CGTAAATCAC TGCATAATTC GTGTCGCTCA AGGCGCACTC CCGTTCTGGA 1800  TAATGTTTTT TGCGCCGACA TCATAACGGT TCTGGCAAAT ATTCTGTTGA CAATTAATCA 1860  TCCGGCTCGT ATAAaGTGTG GAATTGTGAG CGGATAACAA TTTCACACAG GAAACAGCGC 1920  CGCTGAGAAA AAGCGAAGCG GCACTGCTCT TTAACAATTT ATCAGACAAT CTGTGTGGGC 1980  ACTCGACCGG AATTATCGAT TAACTTTATT ATTAAAAATT AAAGAGGTAT ATATTAATGT 2040  ATCGATTAAA TAAGGAGGAA TAAACCATGA CCATTTCCTC ACCTTTGATT GACGTCGCCA 2100  ACCTTCCAGA CATCAACACC ACTGCCGGCA AGATCGCCGA CCTTAAGGCT CGCCGCGCGG 2160 AAGCCCATTT CCCCATGGGT GAAAAGGCAG TAGAGAAGGT CCACGCTGCT GGACGCCTCA 2220 CTGCCCGTGA GCGCTTGGAT TACTTACTCG ATGAGGGCTC CTTCATCGAG ACCGATCAGC 2280 TGGCTCGCCA CCGCACCACC GCTTTCGGCC TGGGCGCTAA GCGTCCTGCA ACCGACGGCA 2340 TCGTGACCGG CTGGGGCACC ATTGATGGAC GCGAAGTCTG CATCTTCTCG CAGGACGGCA 2400  CCGTATTCGG TGGCGCGCTT GGTGAGGTGT ACGGCGAAAA GATGATCAAG ATCATGGAGC 2460  TGGCAATCGA CACCGGCCGC CCATTGATCG GTCTTTACGA AGGCGCTGGC GCTCGTATTC 2520  AGGACGGCGC TGTCTCCCTG GACTTCATTT CCCAGACCTT CTACCAAAAC ATTCAGGCTT 2580  CTGGCGTTAT CCCACAGATC TCCGTCATCA TGGGCGCATG TGCAGGTGGC AACGCTTACG 2640  GCCCAGCTCT GACCGACTTC GTGGTCATGG TGGACAAGAC CTCCAAGATG TTCGTTACCG 2700  GCCCAGACGT GATCAAGACC GTCACCGGCG AGGAAATCAC CCAGGAAGAG CTTGGCGGAG 2760 CAACCACCCA CATGGTGACC GCTGGTAACT CCCACTACAC CGCTGCGACC GATGAGGAAG 2820 CACTGGATTG GGTACAGGAC CTGGTGTCCT TCCTCCCATC CAACAATCGC TCCTACGCAC 2880 CGATGGAAGA CTTCGACGAG GAAGAAGGCG GCGTTGAAGA AAACATCACC GCTGACGATC 2940  TGAAGCTCGA CGAGATCATC CCAGATTCCG CGACCGTTCC TTACGACGTC CGCGATGTCA 3000  TCGAATGCCT CACCGACGAT GGCGAATACC TGGAAATCCA GGCAGACCGC GCAGAAAACG 3060  TTGTTATTGC ATTCGGCCGC ATCGAAGGCC AGTCCGTTGG CTTTGTTGCC AACCAGCCAA 3120  CCCAGTTCGC TGGCTGCCTG GACATCGACT CCTCTGAGAA GGCAGCTCGC TTCGTCCGCA 3180  CCTGCGACGC GTTCAACATC CCAATCGTCA TGCTTGTCGA CGTCCCCGGC TTCCTCCCAG 3240  GCGCAGGCCA GGAGTACGGT GGCATTCTGC GTCGTGGCGC AAAGCTGCTC TACGCATACG 3300 GCGAAGCAAC CGTTCCAAAG ATCACCGTCA CCATGCGTAA GGCTTACGGC GGAGCGTACT 3360 GCGTGATGGG TTCCAAGGGC TTGGGCTCTG ACATCAACCT TGCATGGCCA ACCGCACAGA 3420 TCGCCGTCAT GGGCGCTGCT GGCGCAGTTG GATTCATCTA CCGCAAGGAG CTCATGGCAG 3480 CTGATGCCAA GGGCCTCGAT ACCGTAGCTC TGGCTAAGTC CTTCGAGCGC GAGTATGAAG 3540 ACCACATGCT CAACCCGTAC CACGCTGCAG AACGTGGCCT GATCGACGCC GTGATCCTGC 3600 CAAGCGAAAC CCGCGGACAG ATTTCCCGCA ACCTTCGCCT GCTCAAGCAC AAGAACGTCA 3660 CTCGCCCTGC TCGCAAGCAC GGCAACATGC CACTGTAAgg aggaaaacta aatgtcagtc 3720 gagactcgca agatcaccaa ggttcttgtc gctaaccgtg gtgagattgc aatccgcgtg 3780 ttccgtgcag ctcgagatga aggcatcgga tctgtcgccg tctacgcaga gccagatgca 3840 gatgcaccat tcgtgtcata tgcagacgag gcttttgccc tcggtggcca aacatccgct 3900 gagtcctacc ttgtcattga caagatcatc gatgcggccc gcaagtccgg cgccgacgcc 3960 atccaccccg gctacggctt cctcgcagaa aacgctgact tcgcagaagc agtcatcaac 4020 gaaggcctga tctggattgg accttcacct gagtccatcc gctccctcgg cgacaaggtc 4080 accgctcgcc acatcgcaga taccgccaag gctccaatgg ctcctggcac caaggaacca 4140 gtaaaagacg cagcagaagt tgtggctttc gctgaagaat tcggtctccc aatcgccatc 4200 aaggcagctt tcggtggcgg cggacgtggc atgaaggttg cctacaagat ggaagaagtc 4260 gctgacctct tcgagtccgc aacccgtgaa gcaaccgcag cgttcggccg cggcgagtgc 4320 ttcgtggagc gctacctgga caaggcacgc cacgttgagg ctcaggtcat cgccgataag 4380 cacggcaacg ttgttgtcgc cggaacccgt gactgctccc tgcagcgccg tttccagaag 4440 ctcgtcgaag aagcaccagc accattcccc accgatgacc agcgcgagcg tctccactcc 4500 tccgcgaagg ctatctgtaa ggaagctggc tactacggtg caggcaccgt tgagtacctc 4560 gttggctccg acggcctgat ctccttcctc gaggtcaaca cccgccccca ggtggaacac 4620 ccagtcaccg aagagaccac cggcatcgac ctggtccgcg aaatgttccg catcgcagaa 4680 ggccacgagc tctccatcaa ggaagatcca gctccacgcg gccacgcatt cgagttccgc 4740 atcaacggcg aagacgctgg ctccaacttc atgcctgcac caggcaagat caccagctac 4800 cgcgagccac agggcccagg cgtccgcatg gactccggtg tcgttgaagg ttccgaaatc 4860 tccggacagt tcgactccat gctggcaaag ctgatcgttt ggggcgacac ccgcgagcag 4920 gctctccagc gctcccgccg tgcacttgca gagtacgttg tcgagggcat gccaaccgtt 4980 atcccattcc accagcacat cgtggaaaac ccagcattcg tgggcaacga cgaaggcttc 5040 gagatctaca ccaagtggat cgaagaggtt tgggataacc caatcgcacc ttacgttgac 5100 gcttccgagc tcgacgaaga tgaggacaag accccagcac agaaggttgt tgtggagatc 5160 aacggccgtc gcgttgaggt tgcactccca ggcgatctgg cactcggtgg caccgctggt 5220 cctaagaaga aggccaagaa gcgtcgcgca ggtggtgcaa aggctggcgt atccggcgat 5280 gcagtggcag ctccaatgca gggcactgtc atcaaggtca acgtcgaaga aggcgctgaa 5340 gtcaacgaag gcgacaccgt tgttgtcctc gaggctatga agatggaaaa ccctgtgaag 5400 gctcataagt ccggaaccgt aaccggcctt actgtcgctg caggcgaggg tgtcaacaag 5460 ggcgttgttc tcctcgagat caagtaaTCT AGAGGAGGAA AACTAAATGA ATGTTGACAT 5520 TAGCCGCTCT CGTGAACCGT TGAACGTGGA ACTGTTGAAA GAAAAACTGC TGCAGAACGG 5580 TGATTTCGGT CAAGTGATCT ACGAGAAGGT CACCGGCTCT ACCAATGCGG ACCTGCTGGC 5640 TCTGGCGGGC AGCGGCGCTC CAAACTGGAC CGTCAAGACT GTTGAATTTC AGGACCACGC 5700 CCGTGGCCGT CTGGGTCGTC CGTGGAGCGC ACCGGAGGGT TCCCAAACCA TCGTCAGCGT 5760 TCTGGTCCAA CTGAGCATTG ATCAGGTGGA CCGTATTGGT ACGATCCCGC TGGCCGCAGG 5820 CTTGGCTGTT ATGGATGCGC TGAATGATCT GGGCGTGGAG GGTGCAGGCC TGAAATGGCC 5880 GAACGATGTT CAGATCCACG GTAAGAAGTT GTGCGGTATT CTGGTTGAAG CAACCGGCTT 5940 CGACTCCACT CCGACCGTGG TTATCGGTTG GGGTACGAAT ATCTCGTTGA CGAAAGAAGA 6000 GCTGCCGGTC CCGCACGCGA CCAGCCTGGC CCTGGAGGGT GTTGAAGTTG ACCGTACGAC 6060 GTTCCTGATT AACATGCTGA CCCATCTGCA TACCCGTCTG GATCAGTGGC AGGGTCCGTC 6120 TGTGGACTGG CTGGATGACT ATCGCGCGGT TTGTAGCAGC ATTGGCCAAG ATGTGCGTGT 6180 CCTGCTGCCT GGTGACAAAG AGCTGCTGGG CGAGGCGATT GGCGTGGCGA CCGGTGGTGA 6240 GATCCGTGTG CGCGACGCCA GCGGCACGGT CCACACGCTG AATGCGGGTG AAATCACGCA 6300 TCTGCGTTTG CAATAAAAGC TTGTTTAAAC GGTCTCCAGC TTGGCTGTTT TGGCGGATGA 6360 GAGAAGATTT TCAGCCTGAT ACAGATTAAA TCAGAACGCA GAAGCGGTCT GATAAAACAG 6420 AATTTGCCTG GCGGCAGTAG CGCGGTGGTC CCACCTGACC CCATGCCGAA CTCAGAAGTG 6480 AAACGCCGTA GCGCCGATGG TAGTGTGGGG TCTCCCCATG CGAGAGTAGG GAACTGCCAG 6540 GCATCAAATA AAACGAAAGG CTCAGTCGAA AGACTGGGCC TTTCGTTTTA TCTGTTGTTT 6600 GTCGGTGAAC GCTCTCCTga cGCCTGATGC GGTATTTTCT CCTTACGCAT CTGTGCGGTA 6660 TTTCACACCG CATATGGTGC ACTCTCAGTA CAATCTGCTC TGATGCCGCA TAGTTAAGCC 6720 AGCCCCGACA CCCGCCAACA CCCGCTGACG AGCTTAGTAA AGCCCTCGCT AGATTTTAAT 6780 GCGGATGTTG CGATTACTTC GCCAACTATT GCGATAACAA GAAAAAGCCA GCCTTTCATG 6840 ATATATCTCC CAATTTGTGT AGGGCTTATT ATGCACGCTT AAAAATAATA AAAGCAGACT 6900 TGACCTGATA GTTTGGCTGT GAGCAATTAT GTGCTTAGTG CATCTAACGC TTGAGTTAAG 6960 CCGCGCCGCG AAGCGGCGTC GGCTTGAACG AATTGTTAGA CATTATTTGC CGACTACCTT 7020 GGTGATCTCG CCTTTCACGT AGTGGACAAA TTCTTCCAAC TGATCTGCGC GCGAGGCCAA 7080 GCGATCTTCT TCTTGTCCAA GATAAGCCTG TCTAGCTTCA AGTATGACGG GCTGATACTG 7140 GGCCGGCAGG CGCTCCATTG CCCAGTCGGC AGCGACATCC TTCGGCGCGA TTTTGCCGGT 7200 TACTGCGCTG TACCAAATGC GGGACAACGT AAGCACTACA TTTCGCTCAT CGCCAGCCCA 7260 GTCGGGCGGC GAGTTCCATA GCGTTAAGGT TTCATTTAGC GCCTCAAATA GATCCTGTTC 7320 AGGAACCGGA TCAAAGAGTT CCTCCGCCGC TGGACCTACC AAGGCAACGC TATGTTCTCT 7380 TGCTTTTGTC AGCAAGATAG CCAGATCAAT GTCGATCGTG GCTGGCTCGA AGATACCTGC 7440 AAGAATGTCA TTGCGCTGCC ATTCTCCAAA TTGCAGTTCG CGCTTAGCTG GATAACGCCA 7500 CGGAATGATG TCGTCGTGCA CAACAATGGT GACTTCTACA GCGCGGAGAA TCTCGCTCTC 7560 TCCAGGGGAA GCCGAAGTTT CCAAAAGGTC GTTGATCAAA GCTCGCCGCG TTGTTTCATC 7620 AAGCCTTACG GTCACCGTAA CCAGCAAATC AATATCACTG TGTGGCTTCA GGCCGCCATC 7680 CACTGCGGAG CCGTACAAAT GTACGGCCAG CAACGTCGGT TCGAGATGGC GCTCGATGAC 7740 GCCAACTACC TCTGATAGTT GAGTCGATAC TTCGGCGATC ACCGCTTCCC TCATGATGTT 7800 TAACTTTGTT TTAGGGCGAC TGCCCTGCTG CGTAACATCG TTGCTGCTCC ATAACATCAA 7860 ACATCGACCC ACGGCGTAAC GCGCTTGCTG CTTGGATGCC CGAGGCATAG ACTGTACCCC 7920 AAAAAAACAG TCATAACAAG CCATGAAAAC CGCCACTGCG CCGTTACCAC CGCTGCGTTC 7980 GGTCAAGGTT CTGGACCAGT TGCGTGAGCG CATACGCTAC TTGCATTACA GCTTACGAAC 8040 CGAACAGGCT TATGTCCACT GGGTTCGTGC CTTCATCCGT TTCCACGGTG TGCGTCACCC 8100 GGCAACCTTG GGCAGCAGCG AAGTCGAGGC ATTTCTGTCC TGGCTGGCGA ACGAGCGCAA 8160 GGTTTCGGTC TCCACGCATC GTCAGGCATT GGCGGCCTTG CTGTTCTTCT ACGGCAAGGT 8220 GCTGTGCACG GATCTGCCCT GGCTTCAGGA GATCGGAAGA CCTCGGCCGT CGCGGCGCTT 8280 GCCGGTGGTG CTGACCCCGG ATGAAGTGGTTCGCATCCTCG GTTTTCTGG AAGGCGAGCA 8340 TCGTTTGTTC GCCCAGCTTC TGTATGGAAC GGGCATGCGG ATCAGTGAGG GTTTGCAACT 8400 GCGGGTCAAG GATCTGGATT TCGATCACGG CACGATCATC GTGCGGGAGG GCAAGGGCTC 8460 CAAGGATCGG GCCTTGATGT TACCCGAGAG CTTGGCACCC AGCCTGCGCG AGCAGGGGAA 8520 TTAATTCCCA CGGGTTTTGC TGCCCGCAAA CGGGCTGTTC TGGTGTTGCT AGTTTGTTAT 8580 CAGAATCGCA GATCCGGCTT CAGCCGGTTT GCCGGCTGAA AGCGCTATTT CTTCCAGAAT 8640 TGCCATGATT TTTTCCCCAC GGGAGGCGTC ACTGGCTCCC GTGTTGTCGG CAGCTTTGAT 8700 TCGATAAGCA GCATCGCCTG TTTCAGGCTG TCTATGTGTG ACTGTTGAGC TGTAACAAGT 8760 TGTCTCAGGT GTTCAATTTC ATGTTCTAGT TGCTTTGTTT TACTGGTTTC ACCTGTTCTA 8820 TTAGGTGTTA CATGCTGTTC ATCTGTTACA TTGTCGATCT GTTCATGGTG AACAGCTTTG 8880 AATGCACCAA AAACTCGTAA AAGCTCTGATGTATCTATCTT TTTTACACC GTTTTCATCT 8940 GTGCATATGG ACAGTTTTCC CTTTGATATG TAACGGTGAA CAGTTGTTCT ACTTTTGTTT 9000 GTTAGTCTTG ATGCTTCACT GATAGATACA AGAGCCATAA GAACCTCAGA TCCTTCCGTA 9060 TTTAGCCAGT ATGTTCTCTA GTGTGGTTCG TTGTTTTTGC GTGAGCCATG AGAACGAACC 9120 ATTGAGATCA TACTTACTTT GCATGTCACT CAAAAATTTT GCCTCAAAAC TGGTGAGCTG 9180 AATTTTTGCA GTTAAAGCAT CGTGTAGTGT TTTTCTTAGT CCGTTATGTA GGTAGGAATC 9240 TGATGTAATG GTTGTTGGTA TTTTGTCACC ATTCATTTTT ATCTGGTTGT TCTCAAGTTC 9300 GGTTACGAGA TCCATTTGTC TATCTAGTTC AACTTGGAAA ATCAACGTAT CAGTCGGGCG 9360 GCCTCGCTTA TCAACCACCA ATTTCATATT GCTGTAAGTG TTTAAATCTT TACTTATTGG 9420 TTTCAAAACC CATTGGTTAA GCCTTTTAAA CTCATGGTAG TTATTTTCAA GCATTAACAT 9480 GAACTTAAAT TCATCAAGGC TAATCTCTAT ATTTGCCTTG TGAGTTTTCT TTTGTGTTAG 9540 TTCTTTTAAT AACCACTCAT AAATCCTCAT AGAGTATTTG TTTTCAAAAG ACTTAACATG 9600 TTCCAGATTA TATTTTATGA ATTTTTTTAA CTGGAAAAGA TAAGGCAATA TCTCTTCACT 9660 AAAAACTAAT TCTAATTTTT CGCTTGAGAA CTTGGCATAG TTTGTCCACT GGAAAATCTC 9720 AAAGCCTTTA ACCAAAGGAT TCCTGATTTC CACAGTTCTC GTCATCAGCT CTCTGGTTGC 9780 TTTAGCTAAT ACACCATAAG CATTTTCCCT ACTGATGTTC ATCATCTGAG CGTATTGGTT 9840 ATAAGTGAAC GATACCGTCC GTTCTTTCCT TGTAGGGTTT TCAATCGTGG GGTTGAGTAG 9900  TGCCACACAG CATAAAATTA GCTTGGTTTC ATGCTCCGTT AAGTCATAGC GACTAATCGC 9960  TAGTTCATTT GCTTTGAAAA CAACTAATTC AGACATACAT CTCAATTGGT CTAGGTGATT 10020 TTAAT 10030 63 pDS57 + FH Key Location/Qualifiers accDA1CB + bir FH (pTB.74) FT misc_feature 8066 . . . 8157 circular DNA; FT /note = “lacZalpha” 11469 BP FT /translation = “MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLN FT RLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTI FT CSDAA” FT misc_feature complement(8447 . . . 9457) FT /note = “aadA1- aminoglycoside 3′- adenylyltransferase” FT /translation = “MRSRNWSRTLTERSGGNGAVAVFMACYDCFFGVQSMPRASKQQA FT RYAVGRCLMLWSSNDVTQQGSRPKTKLNIMREAVIAEVSTQLSEVVGVIERHLEPTLL FT AVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVE FT VTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVAL FT VGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIA FT PKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVV FT GK” FT misc_feature complement(10490 . . . 11440) FT /note = “repA protein” FT /translation = “MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRK FT ERTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAK FT FSSEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHK FT ANIEISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTL FT IFQVELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTS FT FEAKFLSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI” FT vector join(1 . . . 329, 8059 . . . 11465) FT /source = “pCL1920revised” FT /type = “Custom cloned vector” FT insert join(330 . . . 1840, 3500 . . . 8061) FT /source = “pCL1920Ptrc” FT /type = “Custom cloned insert” FT misc_feature 7869 . . . 8026 FT /note = “TERM rrnB T1 and T2 transcriptional terminators” FT misc_feature 543 . . . 1625 FT /note = “Lac Repressor lacI ORF” FT /translation = “VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE FT LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS FT GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FT FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG FT DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT FT EDSSCYIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT FT QTASPRALADSLMQLARQVSRLESGQ” FT /note = “Change from C to T” FT modified_base 1644 . . . 1644 FT /note = “Change from G to A” FT insert 1841 . . . 3499 FT /source = “pds57ctg.seq” FT /type = “Custom cloned insert” FT promoter 1854 . . . 2046 FT /note = “Ptrc” FT gene 2077 . . . 3498 FT /note = “ES9(WS377)” FT /translation = “MKRLGTLDASWLAVESEDTPMHVGTLQIFSLPEGAPETFLRDMV FT TRMKEAGDVAPPWGYKLAWSGFLGRVIAPAWKVDKDIDLDYHVRHSALPRPGGERELG FT ILVSRLHSNPLDFSRPLWECHVIEGLENNRFALYTKMHHSMIDGISGVRLMQRVLTTD FT PERCNMPPPWTVRPHQRRGAKTDKEASVPAAVSQAMDALKLQADMAPRLWQAGNRLVH FT SVRHPEDGLTAPFTGPVSVLNHRVTAQRRFATQHYQLDRLKNLAHASGGSLNDIVLYL FT CGTALRRFLAEQNNLPDTPLTAGIPVNIRPADDEGTGTQISFMIASLATDEADPLNRL FT QQIKTSTRRAKEHLQKLPKSALTQYTMLLMSPYILQLMSGLGGRMRPVFNVTISNVPG FT PEGTLYYEGARLEAMYPVSLIAHGGALNITCLSYAGSLNFGFTGCRDTLPSMQKLAVY FT TGEALDELESLILPPKKRARTRK” FT misc_feature 1892 . . . 1912 FT /note = “lac operator” FT −35_signal 1857 . . . 1862 FT /note = “−35” FT −10_signal 1880 . . . 1885 FT /note = “−10” FT misc_feature 1928 . . . 1997 FT /note = “rrnB antitermination” FT RBS 2065 . . . 2069 FT /note = “RBS” FT /note = “Seq-lacI-1” FT gene 3517 . . . 5148 FT /note = “dtsR1(accDA1)” FT /translation = “MTISSPLIDVANLPDINTTAGKIADLKARRAEAHFPMGEKAVEK FT VHAAGRLTARERLDYLLDEGSFIETDQLARHRTTAFGLGAKRPATDGIVTGWGTIDGR FT EVCIFSQDGTVFGGALGEVYGEKMIKIMELAIDTGRPLIGLYEGAGARIQDGAVSLDF FT ISQTFYQNIQASGVIPQISVIMGACAGGNAYGPALTDFVVMVDKTSKMFVTGPDVIKT FT VTGEEITQEELGGATTHMVTAGNSHYTAATDEEALDWVQDLVSFLPSNNRSYAPMEDF FT DEEEGGVEENITADDLKLDEIIPDSATVPYDVRDVIECLTDDGEYLEIQADRAENVVI FT AFGRIEGQSVGFVANQPTQFAGCLDIDSSEKAARFVRTCDAFNIPIVMLVDVPGFLPG FT AGQEYGGILRRGAKLLYAYGEATVPKITVTMRKAYGGAYCVMGSKGLGSDINLAWPTA FT QIAVMGAAGAVGFIYRKELMAADAKGLDTVALAKSFEREYEDHMLNPYHAAERGLIDA FT VILPSETRGQISRNLRLLKHKNVTRPARKHGNMPL” FT gene 5162 . . . 6937 FT /note = “C. glutamicum accCB” FT /translation = “MSVETRKITKVLVANRGEIAIRVFRAARDEGIGSVAVYAEPDAD FT APFVSYADEAFALGGQTSAESYLVIDKIIDAARKSGADAIHPGYGFLAENADFAEAVI FT NEGLIWIGPSPESIRSLGDKVTARHIADTAKAPMAPGTKEPVKDAAEVVAFAEEFGLP FT IAIKAAFGGGGRGMKVAYKMEEVADLFESATREATAAFGRGECFVERYLDKARHVEAQ FT VIADKHGNVVVAGTRDCSLQRRFQKLVEEAPAPFLTDDQRERLHSSAKAICKEAGYYG FT AGTVEYLVGSDGLISFLEVNTRLQVEHPVTEETTGIDLVREMFRIAEGHELSIKEDPA FT PRGHAFEFRINGEDAGSNFMPAPGKITSYREPQGPGVRMDSGVVEGSEISGQFDSMLA FT KLIVWGDTREQALQRSRRALAEYVVEGMPTVIPFHQHIVENPAFVGNDEGFEIYTKWI FT EEVWDNPIAPYVDASELDEDEDKTPAQKVVVEINGRRVEVALPGDLALGGTAGPKKKA FT KKRRAGGAKAGVSGDAVAAPMQGTVIKVNVEEGAEVNEGDTVVVLEAMKMENPVKAHK FT SGTVTGLTVAAGEGVNKGVVLLEIK” FT misc_feature 5177 . . . 5179 FT /note = “rare Arg codon, change to CGT or CGC” FT misc_feature 5162 . . . 5164 FT /note = “GTG start codon, change to ATG” FT gene 6957 . . . 7766 FT /note = “birA_Cg_opt” FT /translation = “MNVDISRSREPLNVELLKEKLLQNGDFGQVIYEKVTGSTNADLL FT ALAGSGAPNWTVKTVEFQDHARGRLGRPWSAPEGSQTIVSVLVQLSIDQVDRIGTIPL FT AAGLAVMDALNDLGVEGAGLKWPNDVQIHGKKLCGILVEATGFDSTPTVVIGWGTNIS FT LTKEELPVPHATSLALEGVEVDRTTFLINMLTHLHTRLDQWQGPSVDWLDDYRAVCSS FT IGQDVRVLLPGDKELLGEAIGVATGGEIRVRDASGTVHTLNAGEITHLRLQ” FT misc_feature 6943 . . . 6948 FT /note = “RBS” FT misc_feature 7771 . . . 7785 FT /dnas_title = “pDS57 + accDA1CBbir” SQ 11469 BP; 2640 A; 3106 C; 3007 G; 2716 T; CACTATACCA ATTGAGATGG GCTAGTCAAT GATAATTACT AGTCCTTTTC CTTTGAGTTG 60 TGGGTATCTG TAAATTCTGC TAGACCTTTG CTGGAAAACT TGTAAATTCT GCTAGACCCT 120 CTGTAAATTC CGCTAGACCT TTGTGTGTTT TTTTTGTTTA TATTCAAGTG GTTATAATTT 180 ATAGAATAAA GAAAGAATAA AAAAAGATAA AAAGAATAGA TCCCAGCCCT GTGTATAACT 240 CACTACTTTA GTCAGTTCCG CAGTATTACA AAAGGATGTC GCAAACGCTG TTTGCTCCTC 300 TACAAAACAG ACCTTAAAAC CCTAAAGGCg tcGGCATCCG CTTACAGACA AGCTGTGACC 360 GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG CGCGAGGCAG 420 CAGATCAATT CGCGCGCGAA GGCGAAGCGG CATGCATTTA CGTTGACACC ATCGAATGGT 480 GCAAAACCTT TCGCGGTATG GCATGATAGC GCCCGGAAGA GAGTCAATTC AGGGTGGTGA 540 ATGTGAAACC AGTAACGTTA TACGATGTCG CAGAGTATGC CGGTGTCTCT TATCAGACCG 600 TTTCCCGCGT GGTGAACCAG GCCAGCCACG TTTCTGCGAA AACGCGGGAA AAAGTGGAAG 660 CGGCGATGGC GGAGCTGAAT TACATTCCCA ACCGCGTGGC ACAACAACTG GCGGGCAAAC 720 AGTCGTTGCT GATTGGCGTT GCCACCTCCA GTCTGGCCCT GCACGCGCCG TCGCAAATTG 780 TCGCGGCGAT TAAATCTCGC GCCGATCAAC TGGGTGCCAG CGTGGTGGTG TCGATGGTAG 840 AACGAAGCGG CGTCGAAGCC TGTAAAGCGG CGGTGCACAA TCTTCTCGCG CAACGCGTCA 900 GTGGGCTGAT CATTAACTAT CCGCTGGATG ACCAGGATGC CATTGCTGTG GAAGCTGCCT 960 GCACTAATGT TCCGGCGTTA TTTCTTGATG TCTCTGACCA GACACCCATC AACAGTATTA 1020  TTTTCTCCCA TGAAGACGGT ACGCGACTGG GCGTGGAGCA TCTGGTCGCA TTGGGTCACC 1080  AGCAAATCGC GCTGTTAGCG GGCCCATTAA GTTCTGTCTC GGCGCGTCTG CGTCTGGCTG 1140  GCTGGCATAA ATATCTCACT CGCAATCAAA TTCAGCCGAT AGCGGAACGG GAAGGCGACT 1200  GGAGTGCCAT GTCCGGTTTT CAACAAACCA TGCAAATGCT GAATGAGGGC ATCGTTCCCA 1260  CTGCGATGCT GGTTGCCAAC GATCAGATGG CGCTGGGCGC AATGCGCGCC ATTACCGAGT 1320 CCGGGCTGCG CGTTGGTGCG GATATCTCGG TAGTGGGATA CGACGATACC GAAGACAGCT 1380 CATGTTATAT CCCGCCGTtA ACCACCATCA AACAGGATTT TCGCCTGCTG GGGCAAACCA 1440 GCGTGGACCG CTTGCTGCAA CTCTCTCAGG GCCAGGCGGT GAAGGGCAAT CAGCTGTTGC 1500 CCGTCTCACT GGTGAAAAGA AAAACCACCC TGGCGCCCAA TACGCAAACC GCCTCTCCCC 1560  GCGCGTTGGC CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC 1620  AGTGAGCGCA ACGCAATTAA TGTaAGTTAG CGCGAATTGA TCTGGTTTGA CAGCTTATCA 1680  TCGACTGCAC GGTGCACCAA TGCTTCTGGC GTCAGGCAGC CATCGGAAGC TGTGGTATGG 1740  CTGTGCAGGT CGTAAATCAC TGCATAATTC GTGTCGCTCA AGGCGCACTC CCGTTCTGGA 1800  TAATGTTTTT TGCGCCGACA TCATAACGGT TCTGGCAAAT ATTCTGAAAT GAGCTGTTGA 1860  CAATTAATCA TCCGGCTCGT ATAATGTGTG GAATTGTGAG CGGATAACAA TTTCACACAG 1920 GAAACAGCGC CGCTGAGAAA AAGCGAAGCG GCACTGCTCT TTAACAATTT ATCAGACAAT 1980  CTGTGTGGGC ACTCGACCGG AATTATCGAT TAACTTTATT ATTAAAAATT AAAGAGGTAT 2040 ATATTAATGT ATCGATTAAA TAAGGAGGAA TAAACCATGA AACGTCTCGG AACCCTGGAC 2100  GCCTCCTGGC TGGCGGTTGA ATCTGAAGAC ACCCCGATGC ATGTGGGTAC GCTTCAGATT 2160  TTCTCACTGC CGGAAGGCGC ACCAGAAACC TTCCTGCGTG ACATGGTCAC TCGAATGAAA 2220  GAGGCCGGCG ATGTGGCACC ACCCTGGGGA TACAAACTGG CCTGGTCTGG TTTCCTCGGG 2280  CGCGTGATCG CCCCGGCCTG GAAAGTCGAT AAGGATATCG ATCTGGATTA TCACGTCCGG 2340  CACTCAGCCC TGCCTCGCCC CGGCGGGGAG CGCGAACTGG GTATTCTGGT ATCCCGACTG 2400  CACTCTAACC CCCTGGATTT TTCCCGCCCT CTTTGGGAAT GCCACGTTAT TGAAGGCCTG 2460 GAGAATAACC GTTTTGCCCT TTACACCAAA ATGCACCACT CGATGATTGA CGGCATCAGC 2520 GGCGTGCGAC TGATGCAGAG GGTGCTCACC ACCGATCCCG AACGCTGCAATATGCCACCG 2580 CCCTGGACGG TACGCCCACA CCAACGCCGT GGTGCAAAAA CCGACAAAGA GGCCAGCGTG 2640 CCCGCAGCGG TTTCCCAGGC AATGGACGCC CTGAAGCTCC AGGCAGACAT GGCCCCCAGG 2700  CTGTGGCAGG CCGGCAATCG CCTGGTGCAT TCGGTTCGAC ACCCGGAAGA CGGACTGACC 2760  GCGCCCTTCA CTGGACCGGT TTCGGTGCTC AATCACCGGG TTACCGCGCA GCGACGTTTT 2820  GCCACCCAGC ATTATCAACT GGACCGGCTG AAAAACCTGG CCCATGCTTC CGGCGGTTCC 2880  TTGAACGACA TCGTGCTTTA CCTGTGTGGC ACCGCATTGC GGCGCTTTCT GGCTGAGCAG 2940 AACAATCTGC CAGACACCCC GCTGACGGCT GGTATACCG GTGAATATCCG GCCGGCAGAC 3000 GACGAGGGTA CGGGCACCCA GATCAGTTTT ATGATTGCCT CGCTGGCCAC CGACGAAGCT 3060 GATCCGTTGA ACCGCCTGCA ACAGATCAAA ACCTCGACCC GACGGGCCAA GGAGCACCTG 3120  CAGAAACTTC CAAAAAGTGC CCTGACCCAG TACACCATGC TGCTGATGTC ACCCTACATT 3180  CTGCAATTGA TGTCAGGTCT CGGGGGGAGG ATGCGACCAG TCTTCAACGT GACCATTTCC 3240  AACGTGCCCG GCCCGGAAGG CACGCTGTAT TATGAAGGAG CCCGGCTTGA GGCCATGTAT 3300  CCGGTATCGC TAATCGCTCA CGGCGGCGCC CTGAACATCA CCTGCCTGAG CTATGCCGGA 3360  TCGCTGAATT TCGGTTTTAC CGGCTGTCGG GATACGCTGC CGAGCATGCA GAAACTGGCG 3420  GTTTATACCG GTGAAGCTCT GGATGAGCTG GAATCGCTGA TTCTGCCACC CAAGAAGCGC 3480 GCCCGAACCC GCAAGTAACT CGAggaggaa aactaaATGA CCATTTCCTC ACCTTTGATT 3540 GACGTCGCCA ACCTTCCAGA CATCAACACC ACTGCCGGCA AGATCGCCGA CCTTAAGGCT 3600 CGCCGCGCGG AAGCCCATTT CCCCATGGGT GAAAAGGCAG TAGAGAAGGT CCACGCTGCT 3660 GGACGCCTCA CTGCCCGTGA GCGCTTGGAT TACTTACTCG ATGAGGGCTC CTTCATCGAG 3720  ACCGATCAGC TGGCTCGCCA CCGCACCACC GCTTTCGGCC TGGGCGCTAA GCGTCCTGCA 3780  ACCGACGGCA TCGTGACCGG CTGGGGCACC ATTGATGGAC GCGAAGTCTG CATCTTCTCG 3840  CAGGACGGCA CCGTATTCGG TGGCGCGCTT GGTGAGGTGT ACGGCGAAAA GATGATCAAG 3900  ATCATGGAGC TGGCAATCGA CACCGGCCGC CCATTGATCG GTCTTTACGA AGGCGCTGGC 3960  GCTCGTATTC AGGACGGCGC TGTCTCCCTG GACTTCATTT CCCAGACCTT CTACCAAAAC 4020  ATTCAGGCTT CTGGCGTTAT CCCACAGATC TCCGTCATCA TGGGCGCATG TGCAGGTGGC 4080 AACGCTTACG GCCCAGCTCT GACCGACTTC GTGGTCATGG TGGACAAGAC CTCCAAGATG 4140  TTCGTTACCG GCCCAGACGT GATCAAGACC GTCACCGGCG AGGAAATCAC CCAGGAAGAG 4200 CTTGGCGGAG CAACCACCCA CATGGTGACC GCTGGTAACT CCCACTACAC CGCTGCGACC 4260  GATGAGGAAG CACTGGATTG GGTACAGGAC CTGGTGTCCT TCCTCCCATC CAACAATCGC 4320  TCCTACGCAC CGATGGAAGA CTTCGACGAG GAAGAAGGCG GCGTTGAAGA AAACATCACC 4380  GCTGACGATC TGAAGCTCGA CGAGATCATC CCAGATTCCG CGACCGTTCC TTACGACGTC 4440  CGCGATGTCA TCGAATGCCT CACCGACGAT GGCGAATACC TGGAAATCCA GGCAGACCGC 4500  GCAGAAAACG TTGTTATTGC ATTCGGCCGC ATCGAAGGCC AGTCCGTTGG CTTTGTTGCC 4560  AACCAGCCAA CCCAGTTCGC TGGCTGCCTG GACATCGACT CCTCTGAGAA GGCAGCTCGC 4620 TTCGTCCGCA CCTGCGACGC GTTCAACATC CCAATCGTCA TGCTTGTCGA CGTCCCCGGC 4680 TTCCTCCCAG GCGCAGGCCA GGAGTACGGT GGCATTCTGC GTCGTGGCGC AAAGCTGCTC 4740 TACGCATACG GCGAAGCAAC CGTTCCAAAG ATCACCGTCA CCATGCGTAA GGCTTACGGC 4800 GGAGCGTACT GCGTGATGGG TTCCAAGGGC TTGGGCTCTG ACATCAACCT TGCATGGCCA 4860  ACCGCACAGA TCGCCGTCAT GGGCGCTGCT GGCGCAGTTG GATTCATCTA CCGCAAGGAG 4920 CTCATGGCAG CTGATGCCAA GGGCCTCGAT ACCGTAGCTC TGGCTAAGTC CTTCGAGCGC 4980 GAGTATGAAG ACCACATGCT CAACCCGTAC CACGCTGCAG AACGTGGCCT GATCGACGCC 5040 GTGATCCTGC CAAGCGAAAC CCGCGGACAG ATTTCCCGCA ACCTTCGCCT GCTCAAGCAC 5100 AAGAACGTCA CTCGCCCTGC TCGCAAGCAC GGCAACATGC CACTGTAAgg aggaaaacta 5160 aatgtcagtc gagactcgca agatcaccaa ggttcttgtc gctaaccgtg gtgagattgc 5220 aatccgcgtg ttccgtgcag ctcgagatga aggcatcgga tctgtcgccg tctacgcaga 5280  gccagatgca gatgcaccat tcgtgtcata tgcagacgag gcttttgccc tcggtggcca 5340 aacatccgct gagtcctacc ttgtcattga caagatcatc gatgcggccc gcaagtccgg 5400 cgccgacgcc atccaccccg gctacggctt cctcgcagaa aacgctgact tcgcagaagc 5460 agtcatcaac gaaggcctga tctggattgg accttcacct gagtccatcc gctccctcgg 5520 cgacaaggtc accgctcgcc acatcgcaga taccgccaag gctccaatgg ctcctggcac 5580 caaggaacca gtaaaagacg cagcagaagt tgtggctttc gctgaagaat tcggtctccc 5640 aatcgccatc aaggcagctt tcggtggcgg cggacgtggc atgaaggttg cctacaagat 5700  ggaagaagtc gctgacctct tcgagtccgc aacccgtgaa gcaaccgcag cgttcggccg 5760 cggcgagtgc ttcgtggagc gctacctgga caaggcacgc cacgttgagg ctcaggtcat 5820 cgccgataag cacggcaacg ttgttgtcgc cggaacccgt gactgctccc tgcagcgccg 5880 tttccagaag ctcgtcgaag aagcaccagc accattcctc accgatgacc agcgcgagcg 5940 tctccactcc tccgcgaagg ctatctgtaa ggaagctggc tactacggtg caggcaccgt 6000 tgagtacctc gttggctccg acggcctgat ctccttcctc gaggtcaaca cccgcctcca 6060 ggtggaacac ccagtcaccg aagagaccac cggcatcgac ctggtccgcg aaatgttccg 6120 catcgcagaa ggccacgagc tctccatcaa ggaagatcca gctccacgcg gccacgcatt 6180 cgagttccgc atcaacggcg aagacgctgg ctccaacttc atgcctgcac caggcaagat 6240 caccagctac cgcgagccac agggcccagg cgtccgcatg gactccggtg tcgttgaagg 6300 ttccgaaatc tccggacagt tcgactccat gctggcaaag ctgatcgttt ggggcgacac 6360 ccgcgagcag gctctccagc gctcccgccg tgcacttgca gagtacgttg tcgagggcat 6420 gccaaccgtt atcccattcc accagcacat cgtggaaaac ccagcattcg tgggcaacga 6480 cgaaggcttc gagatctaca ccaagtggat cgaagaggtt tgggataacc caatcgcacc 6540 ttacgttgac gcttccgagc tcgacgaaga tgaggacaag accccagcac agaaggttgt 6600 tgtggagatc aacggccgtc gcgttgaggt tgcactccca ggcgatctgg cactcggtgg 6660 caccgctggt cctaagaaga aggccaagaa gcgtcgcgca ggtggtgcaa aggctggcgt 6720 atccggcgat gcagtggcag ctccaatgca gggcactgtc atcaaggtca acgtcgaaga 6780 aggcgctgaa gtcaacgaag gcgacaccgt tgttgtcctc gaggctatga agatggaaaa 6840 ccctgtgaag gctcataagt ccggaaccgt aaccggcctt actgtcgctg caggcgaggg 6900 tgtcaacaag ggcgttgttc tcctcgagat caagtaaTCT AGAGGAGGAA AACTAAATGA 6960 ATGTTGACAT TAGCCGCTCT CGTGAACCGT TGAACGTGGA ACTGTTGAAA GAAAAACTGC 7020 TGCAGAACGG TGATTTCGGT CAAGTGATCT ACGAGAAGGT CACCGGCTCT ACCAATGCGG 7080  ACCTGCTGGC TCTGGCGGGC AGCGGCGCTC CAAACTGGAC CGTCAAGACT GTTGAATTTC 7140  AGGACCACGC CCGTGGCCGT CTGGGTCGTC CGTGGAGCGC ACCGGAGGGT TCCCAAACCA 7200  TCGTCAGCGT TCTGGTCCAA CTGAGCATTG ATCAGGTGGA CCGTATTGGT ACGATCCCGC 7260 TGGCCGCAGG CTTGGCTGTT ATGGATGCGC TGAATGATCT GGGCGTGGAG GGTGCAGGCC 7320 TGAAATGGCC GAACGATGTT CAGATCCACG GTAAGAAGTT GTGCGGTATT CTGGTTGAAG 7380 CAACCGGCTT CGACTCCACT CCGACCGTGG TTATCGGTTG GGGTACGAAT ATCTCGTTGA 7440  CGAAAGAAGA GCTGCCGGTC CCGCACGCGA CCAGCCTGGC CCTGGAGGGT GTTGAAGTTG 7500  ACCGTACGAC GTTCCTGATT AACATGCTGA CCCATCTGCA TACCCGTCTG GATCAGTGGC 7560  AGGGTCCGTC TGTGGACTGG CTGGATGACT ATCGCGCGGT TTGTAGCAGC ATTGGCCAAG 7620  ATGTGCGTGT CCTGCTGCCT GGTGACAAAG AGCTGCTGGG CGAGGCGATT GGCGTGGCGA 7680  CCGGTGGTGA GATCCGTGTG CGCGACGCCA GCGGCACGGT CCACACGCTG AATGCGGGTG 7740  AAATCACGCA TCTGCGTTTG CAATAAGTTT AAACGGTCTC CAGCTTGGCT GTTTTGGCGG 7800 ATGAGAGAAG ATTTTCAGCC TGATACAGAT TAAATCAGAA CGCAGAAGCG GTCTGATAAA 7860 ACAGAATTTG CCTGGCGGCA GTAGCGCGGT GGTCCCACCT GACCCCATGC CGAACTCAGA 7920 AGTGAAACGC CGTAGCGCCG ATGGTAGTGT GGGGTCTCCC CATGCGAGAG TAGGGAACTG 7980 CCAGGCATCA AATAAAACGA AAGGCTCAGT CGAAAGACTG GGCCTTTCGT TTTATCTGTT 8040  GTTTGTCGGT GAACGCTCTC CTgacGCCTG ATGCGGTATT TTCTCCTTAC GCATCTGTGC 8100  GGTATTTCAC ACCGCATATG GTGCACTCTC AGTACAATCT GCTCTGATGC CGCATAGTTA 8160  AGCCAGCCCC GACACCCGCC AACACCCGCT GACGAGCTTA GTAAAGCCCT CGCTAGATTT 8220  TAATGCGGAT GTTGCGATTA CTTCGCCAAC TATTGCGATA ACAAGAAAAA GCCAGCCTTT 8280  CATGATATAT CTCCCAATTT GTGTAGGGCT TATTATGCAC GCTTAAAAAT AATAAAAGCA 8340  GACTTGACCT GATAGTTTGG CTGTGAGCAA TTATGTGCTT AGTGCATCTA ACGCTTGAGT 8400 TAAGCCGCGC CGCGAAGCGG CGTCGGCTTG AACGAATTGT TAGACATTAT TTGCCGACTA 8460  CCTTGGTGAT CTCGCCTTTC ACGTAGTGGA CAAATTCTTC CAACTGATCT GCGCGCGAGG 8520 CCAAGCGATC TTCTTCTTGT CCAAGATAAG CCTGTCTAGC TTCAAGTATG ACGGGCTGAT 8580  ACTGGGCCGG CAGGCGCTCC ATTGCCCAGT CGGCAGCGAC ATCCTTCGGC GCGATTTTGC 8640  CGGTTACTGC GCTGTACCAA ATGCGGGACA ACGTAAGCAC TACATTTCGC TCATCGCCAG 8700  CCCAGTCGGG CGGCGAGTTC CATAGCGTTA AGGTTTCATT TAGCGCCTCA AATAGATCCT 8760  GTTCAGGAAC CGGATCAAAG AGTTCCTCCG CCGCTGGACC TACCAAGGCA ACGCTATGTT 8820  CTCTTGCTTT TGTCAGCAAG ATAGCCAGAT CAATGTCGAT CGTGGCTGGC TCGAAGATAC 8880  CTGCAAGAAT GTCATTGCGC TGCCATTCTC CAAATTGCAG TTCGCGCTTA GCTGGATAAC 8940 GCCACGGAAT GATGTCGTCG TGCACAACAA TGGTGACTTC TACAGCGCGG AGAATCTCGC 9000 TCTCTCCAGG GGAAGCCGAA GTTTCCAAAA GGTCGTTGAT CAAAGCTCGC CGCGTTGTTT 9060 CATCAAGCCT TACGGTCACC GTAACCAGCA AATCAATATC ACTGTGTGGC TTCAGGCCGC 9120 CATCCACTGC GGAGCCGTAC AAATGTACGG CCAGCAACGT CGGTTCGAGA TGGCGCTCGA 9180  TGACGCCAAC TACCTCTGAT AGTTGAGTCG ATACTTCGGC GATCACCGCT TCCCTCATGA 9240  TGTTTAACTT TGTTTTAGGG CGACTGCCCT GCTGCGTAAC ATCGTTGCTG CTCCATAACA 9300  TCAAACATCG ACCCACGGCG TAACGCGCTT GCTGCTTGGA TGCCCGAGGC ATAGACTGTA 9360  CCCCAAAAAA ACAGTCATAA CAAGCCATGA AAACCGCCAC TGCGCCGTTA CCACCGCTGC 9420 GTTCGGTCAA GGTTCTGGAC CAGTTGCGTG AGCGCATACG CTACTTGCAT TACAGCTTAC 9480 GAACCGAACA GGCTTATGTC CACTGGGTTC GTGCCTTCAT CCGTTTCCAC GGTGTGCGTC 9540 ACCCGGCAAC CTTGGGCAGC AGCGAAGTCG AGGCATTTCT GTCCTGGCTG GCGAACGAGC 9600  GCAAGGTTTC GGTCTCCACG CATCGTCAGG CATTGGCGGC CTTGCTGTTC TTCTACGGCA 9660  AGGTGCTGTG CACGGATCTG CCCTGGCTTC AGGAGATCGG AAGACCTCGG CCGTCGCGGC 9720  GCTTGCCGGT GGTGCTGACC CCGGATGAAG TGGTTCGCAT CCTCGGTTTT CTGGAAGGCG 9780  AGCATCGTTT GTTCGCCCAG CTTCTGTATG GAACGGGCAT GCGGATCAGT GAGGGTTTGC 9840  AACTGCGGGT CAAGGATCTG GATTTCGATC ACGGCACGAT CATCGTGCGG GAGGGCAAGG 9900  GCTCCAAGGA TCGGGCCTTG ATGTTACCCG AGAGCTTGGC ACCCAGCCTG CGCGAGCAGG 9960 GGAATTAATT CCCACGGGTT TTGCTGCCCG CAAACGGGCT GTTCTGGTGT TGCTAGTTTG 10020 TTATCAGAAT CGCAGATCCG GCTTCAGCCG GTTTGCCGGC TGAAAGCGCT ATTTCTTCCA 10080 GAATTGCCAT GATTTTTTCC CCACGGGAGG CGTCACTGGC TCCCGTGTTG TCGGCAGCTT 10140 TGATTCGATA AGCAGCATCG CCTGTTTCAG GCTGTCTATG TGTGACTGTT GAGCTGTAAC 10200 AAGTTGTCTC AGGTGTTCAA TTTCATGTTC TAGTTGCTTT GTTTTACTGG TTTCACCTGT 10260 TCTATTAGGT GTTACATGCT GTTCATCTGT TACATTGTCG ATCTGTTCAT GGTGAACAGC 10320 TTTGAATGCA CCAAAAACTC GTAAAAGCTC TGATGTATCT ATCTTTTTTA CACCGTTTTC 10380 ATCTGTGCAT ATGGACAGTT TTCCCTTTGA TATGTAACGG TGAACAGTTG TTCTACTTTT 10440 GTTTGTTAGT CTTGATGCTT CACTGATAGA TACAAGAGCC ATAAGAACCT CAGATCCTTC 10500 CGTATTTAGC CAGTATGTTC TCTAGTGTGG TTCGTTGTTT TTGCGTGAGC CATGAGAACG 10560 AACCATTGAG ATCATACTTA CTTTGCATGT CACTCAAAAA TTTTGCCTCA AAACTGGTGA 10620 GCTGAATTTT TGCAGTTAAA GCATCGTGTA GTGTTTTTCT TAGTCCGTTA TGTAGGTAGG 10680 AATCTGATGT AATGGTTGTT GGTATTTTGT CACCATTCAT TTTTATCTGG TTGTTCTCAA 10740 GTTCGGTTAC GAGATCCATT TGTCTATCTA GTTCAACTTG GAAAATCAAC GTATCAGTCG 10800 GGCGGCCTCG CTTATCAACC ACCAATTTCA TATTGCTGTA AGTGTTTAAA TCTTTACTTA 10860 TTGGTTTCAA AACCCATTGG TTAAGCCTTT TAAACTCATG GTAGTTATTT TCAAGCATTA 10920 ACATGAACTT AAATTCATCA AGGCTAATCT CTATATTTGC CTTGTGAGTT TTCTTTTGTG 10980 TTAGTTCTTT TAATAACCAC TCATAAATCC TCATAGAGTA TTTGTTTTCA AAAGACTTAA 11040 CATGTTCCAG ATTATATTTT ATGAATTTTT TTAACTGGAA AAGATAAGGC AATATCTCTT 11100 CACTAAAAAC TAATTCTAAT TTTTCGCTTG AGAACTTGGC ATAGTTTGTC CACTGGAAAA 11160 TCTCAAAGCC TTTAACCAAA GGATTCCTGA TTTCCACAGT TCTCGTCATC AGCTCTCTGG 11220 TTGCTTTAGC TAATACACCA TAAGCATTTT CCCTACTGAT GTTCATCATC TGAGCGTATT 11280 GGTTATAAGT GAACGATACC GTCCGTTCTT TCCTTGTAGG GTTTTCAATC GTGGGGTTGA 11340 GTAGTGCCAC ACAGCATAAA ATTAGCTTGG TTTCATGCTC CGTTAAGTCA TAGCGACTAA 11400 TCGCTAGTTC ATTTGCTTTG AAAACAACTA ATTCAGACAT ACATCTCAAT TGGTCTAGGT 11460 GATTTTAAT 11470 

What is claimed is:
 1. A cultured genetically engineered host cell comprising: (a) a polynucleotide sequence encoding one or more of: (i) an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, (iv) a sequence having a transposon insertion in the yijP gene, and (v) a heterologous ACP protein; and (b) a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide, wherein the genetically engineered host cell produces a fatty acid derivative composition at a higher titer, yield or productivity when cultured in medium containing a carbon source under conditions effective to overexpress the polynucleotide(s) relative to a corresponding wild type host cell propagated under the same conditions as the genetically engineered host cell.
 2. The genetically engineered host cell of claim 1, wherein the fatty acid derivative composition comprises a fatty acid derivative selected from the group consisting of a fatty acid, a fatty aldehyde, a fatty alcohol, a fatty ester, an alkane, a terminal olefin, an internal olefin and a ketone.
 3. The genetically engineered host cell of claim 1 or 2, wherein the fatty acid derivative composition is produced at a titer that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the titer of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell.
 4. The genetically engineered host cell of claim 1 or 2, wherein the fatty acid derivative composition is produced at a titer of at least 100 mg/L.
 5. The genetically engineered host cell of claim 1 or 2, wherein the fatty acid derivative composition is produced at a titer of from 30 g/L to 250 g/L.
 6. The genetically engineered host cell of claim 1 or 2, wherein the fatty acid derivative composition is produced at a yield that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the yield of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell.
 7. The genetically engineered host cell of claim 1 or 2, wherein the fatty acid derivative composition has a yield of from 10% to 40%.
 8. The genetically engineered host cell of claim 1 or 2, wherein the fatty acid derivative composition is produced at a productivity that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the productivity of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell.
 9. The genetically engineered host cell of claim 1 or 2, wherein the fatty acid derivative composition is produced at a productivity of from 0.7 mg/L/hr to 3 g/L/hr.
 10. The genetically engineered host cell of claim 1 or 2, wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is overexpressed.
 11. The genetically engineered host cell of claim 10, wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is accD+.
 12. The genetically engineered host cell of any one of claims 1 to 11, wherein the FadR polypeptide is overexpressed.
 13. The genetically engineered host cell of any one of claims 1 to 12, wherein the heterologous iFAB polypeptide is overexpressed.
 14. The genetically engineered host cell of claim 13, wherein the heterologous iFAB polypeptide is iFAB
 138. 15. The genetically engineered host cell of any one of claims 1 to 14, wherein the host cell comprises a transposon insertion in the yijP gene.
 16. The genetically engineered host cell of any one of claims 1 to 15, wherein the host cell comprises a heterologous acp sequence.
 17. The genetically engineered host cell of claim 16, further comprising an sfp gene.
 18. The genetically engineered host cell of any one of claims 1 to 17, wherein the polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide is selected from the group consisting of a polypeptide: (a) having thioesterase activity, wherein the recombinant host cell synthesizes fatty acids; (b) having thioesterase activity and carboxylic acid reductase (“CAR”) activity, wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; (c) having thioesterase activity, carboxylic acid reductase activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; (d) having acyl-CoA reductase (“AAR”) activity wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; (e) having acyl-CoA reductase (“AAR”) activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; (f) having fatty alcohol forming acyl-CoA reductase (“FAR”) activity, wherein the recombinant host cell synthesizes fatty alcohols; (g) having thioesterase activity, carboxylic acid reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; (h) having acyl-CoA reductase (“AAR”) activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; (i) having ester synthase activity wherein the recombinant host cell synthesizes fatty esters; (j) having thioesterase activity, acyl-CoA synthase activity and ester synthase activity wherein the recombinant host cell synthesizes fatty esters; (k) having OleA activity, wherein the recombinant host cell synthesizes aliphatic ketones; (l) having OleABCD activity, wherein the recombinant host cell synthesizes internal olefins; and (m) having thioesterase activity and decarboxylase activity, wherein the recombinant host cell synthesizes terminal olefins.
 19. The genetically engineered host cell of any one of claims 1 to 16, wherein the fatty acid derivative composition is produced extracellularly.
 20. A cell culture comprising the genetically engineered host cell of any one of claims 1 to
 19. 21. The cell culture of claim 20, wherein the culture medium comprises a fatty acid derivative composition.
 22. The cell culture of claim 20, wherein the fatty acid derivative composition comprises at least one fatty acid derivative selected from the group consisting of a fatty acid, a fatty aldehyde, a fatty alcohol, a fatty ester, an alkane, a terminal olefin, an internal olefin and a ketone.
 23. The cell culture of claim 20, wherein the fatty acid derivative is a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty acid derivative.
 24. The cell culture of claim 20, wherein the fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative.
 25. The cell culture of claim 20, wherein the fatty acid derivative composition comprises one or more of C₈, C₁₀, C₁₂, C₁₄, C₁₆, and C₁₈ fatty acid derivatives.
 26. The cell culture of claim 22, wherein the fatty acid derivative composition comprises fatty acids.
 27. The cell culture of claim 22, wherein the fatty acid derivative composition comprises fatty aldehydes.
 28. The cell culture of claim 22, wherein the fatty acid derivative composition comprises fatty alcohols.
 29. The cell culture of claim 22, wherein the fatty acid derivative composition comprises fatty esters.
 30. The cell culture of claim 22, wherein the fatty acid derivative composition comprises an alkane.
 31. The cell culture of claim 22, wherein the fatty acid derivative composition comprises a terminal olefin.
 32. The cell culture of claim 22, wherein the fatty acid derivative composition comprises an internal olefin.
 33. The cell culture of claim 22, wherein the fatty acid derivative composition comprises a ketone.
 34. The cell culture of claim 20, wherein the fatty acid derivative composition comprises a fatty acid derivative having a double bond at position 7 in the carbon chain (between C₇ and C₈) from the reduced end of the fatty alcohol.
 35. The cell culture of claim 20, wherein the fatty acid derivative composition comprises unsaturated fatty acid derivatives.
 36. The cell culture of claim 20, wherein the fatty acid derivative composition comprises saturated fatty acid derivatives.
 37. The cell culture of claim 20, wherein the fatty acid derivative composition comprises branched chain fatty acid derivatives.
 38. The cell culture of claim 20, wherein the fatty acid derivative has a fraction of modern carbon of about 1.003 to about 1.5.
 39. The cell culture of claim 20, wherein the fatty acid derivative has a δ¹³C of from about −10.9 to about −15.4.
 40. A cultured recombinant host cell, engineered to increase the production of malonyl CoA, comprising: a polynucleotide sequence encoding one or more of: (i) an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, or (iv) a sequence having a transposon insertion in the yijP gene, wherein the engineered host cells produces a fatty acid derivative composition at a higher titer, yield or productivity when cultured in medium containing a carbon source under conditions effective to overexpress the polynucleotide(s) relative to a corresponding wild type host cell propagated under the same conditions as the genetically engineered host cell.
 41. The cultured recombinant host cell of claim 40, wherein the host cell further comprises a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide.
 42. The cultured recombinant host cell of claim 40, wherein the fatty acid derivative composition produced by the cultured genetically engineered host cell has a titer that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the titer of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell.
 43. The cultured recombinant host cell of claim 40, wherein the host cell has a titer of at least 100 mg/L.
 44. The cultured recombinant host cell of claim 40, wherein the fatty acid derivative composition produced by the cultured genetically engineered host cell has a titer of from 30 g/L to 250 g/L.
 45. The cultured recombinant host cell of claim 40, wherein the fatty acid derivative composition produced by a cultured genetically engineered host cell has a yield that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the yield of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell.
 46. The cultured recombinant host cell of claim 40, wherein the fatty acid derivative composition produced by the cultured genetically engineered host cell has a yield of from 10% to 40%.
 47. The cultured recombinant host cell of claim 40, wherein the fatty acid derivative composition produced by a cultured genetically engineered host cell has a productivity that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the productivity of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell.
 48. The cultured recombinant host cell of claim 40, wherein the fatty acid derivative composition produced by a cultured genetically engineered host cell has a productivity of from 0.7 mg/L/hr to 3 g/L/hr.
 49. The cultured recombinant host cell of any one of claims 40 to 49, wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is overexpressed.
 50. The cultured recombinant host cell of claim 49, wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is accD+.
 51. The cultured recombinant host cell of any one of claims 40 to 50, wherein the FadR polypeptide is overexpressed.
 52. The cultured recombinant host cell of any one of claims 40 to 51, wherein the heterologous iFAB polypeptide is overexpressed.
 53. The cultured recombinant host cell of claim 52 wherein the heterologous iFAB polypeptide is iFAB
 138. 54. The cultured recombinant host cell of any one of claims 40 to 53, wherein the host cell comprises a transposon insertion in the yijP gene.
 55. A cell culture comprising the cultured recombinant host cell of any one of claims 40 to 54
 56. A method of making a fatty acid derivative composition, comprising the steps of: (a) engineering a parental host cell to obtain a recombinant host cell which comprises an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, and (iv) a sequence having a transposon insertion in the yijP gene; (b) further engineering the cell to comprise polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide; (c) culturing the recombinant host cell in the presence of a carbon source under conditions effective to result in a yield, titer or productivity of the fatty acid derivative composition that is at least 3 times the yield, titer or productivity of fatty acid derivative composition produced by the parental microbial cell cultured under the same conditions; and (d) optionally isolating the fatty acid derivative composition.
 57. The method of claim 56, wherein the host cell is further engineered to comprise a polynucleotide sequence encoding a heterologous acp protein.
 58. The method of claim 57, wherein the host cell is further engineered to comprise an sfp gene.
 59. The method of claim 56, wherein the fatty acid derivative biosynthetic polypeptide is selected from the group consisting of a polypeptide: (a) having thioesterase activity, wherein the recombinant host cell synthesizes fatty acids; (b) having thioesterase activity and carboxylic acid reductase (“CAR”) activity, wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; (c) having thioesterase activity, carboxylic acid reductase activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; (d) having acyl-CoA reductase (“AAR”) activity wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; (e) having acyl-CoA reductase (“AAR”) activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; (f) having fatty alcohol forming acyl-CoA reductase (“FAR”) activity, wherein the recombinant host cell synthesizes fatty alcohols; (g) having thioesterase activity, carboxylic acid reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; (h) having acyl-CoA reductase (“AAR”) activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; (i) having ester synthase activity wherein the recombinant host cell synthesizes fatty esters; (j) having thioesterase activity, acyl-CoA synthase activity and ester synthase activity wherein the recombinant host cell synthesizes fatty esters; (k) having OleA activity, wherein the recombinant host cell synthesizes aliphatic ketones; (l) having OleABCD activity, wherein the recombinant host cell synthesizes internal olefins; and (m) having thioesterase activity and decarboxylase activity, wherein the recombinant host cell synthesizes terminal olefins.
 60. The method of claim 56, where in the fatty acid derivative is selected from the group consisting of a fatty acid, a fatty alcohol, a fatty aldehyde, a fatty acid ester, a hydrocarbon, a ketone, and an olefin.
 61. The method of claim 56, where in the fatty acid derivative is a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty acid derivative.
 62. The method of claim 56, where in the fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative.
 63. A method of making a fatty acid derivative composition with a higher titer, yield or productivity of fatty acid derivatives than produced by a parental host cell, the method comprising: (a) engineering a parental host cell to obtain a recombinant host cell which comprises one or more of: (i) a polynucleotide encoding an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a polynucleotide encoding a FadR polypeptide, (iii) a polynucleotide encoding a heterologous iFAB polypeptide, (iv) a sequence having a transposon insertion in the yijP gene, and (v) a polynucleotide encoding a heterologous ACP protein; (b) further engineering the cell to comprise a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide; (c) culturing the recombinant host cell in the presence of a carbon source under conditions effective to result in a yield, titer or productivity of fatty acid derivatives that is at least 3 times the yield, titer or productivity of fatty acid derivatives produced by the parental microbial cell cultured under the same conditions; and (d) optionally isolating the fatty acid derivative composition. 